Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients.

Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separation dimension (2D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS. This study reports proof-of-principle results of the development of an RPLC × RPLC-HRMS method using parallel gradients (2D flow rate of 0.7 mL min-1) and its comparison to shifted gradient methods (2D of 1.4 mL min-1) for the analysis of complex digests using HRMS (QExactive-Plus MS). Shifted and parallel gradients resulted in high surface coverage (SC) and effective peak capacity (SC of 0.6226 and 0.7439 and effective peak capacity of 779 and 757 in 60 min). When applied to a cell line digest sample, parallel gradients allowed higher sensitivity (e.g., average MS intensity increased by a factor of 3), allowing for a higher number of identifications (e.g., about 2600 vs 3900 peptides). In addition, reducing the modulation time to 10 s significantly increased the number of MS/MS events that could be performed. When compared to a 1D-RPLC method, parallel RPLC × RPLC-HRMS methods offered a higher separation performance (FHWH from 0.12 to 0.018 min) with limited sensitivity losses resulting in an increase of analyte identifications (e.g., about 6000 vs 7000 peptides and 1500 vs 1990 proteins).

Tunable Gas Admission via a "Molecular Trapdoor" Mechanism in a Flexible Cationic Metal-Organic Framework Featuring 1D Channels.

Achieving high gas selectivity is challenging when dealing with gas pairs of similar size and physiochemical properties. The "molecular trapdoor" mechanism discovered in zeolites holds promise for highly selective gas adsorption separation but faces limitations like constrained pore volume and slow adsorption kinetics. To address these challenges, for the first time, a flexible metal-organic framework (MOF) featuring 1D channels and functioning as a "molecular trapdoor" material is intoduced. Extra-framework anions act as "gate-keeping" groups at the narrowest points of channels, permitting gas admissions via gate opening induced by thermal/pressure stimuli and guest interactions. Different guest molecules induce varied energy barriers for anion movement, enabling gas separation based on distinct threshold temperatures for gas admission. The flexible framework of Pytpy MOFs, featuring swelling structure with rotatable pyridine rings, facilitates faster gas adsorption than zeolite. Analyzing anion properties of Pytpy MOFs reveals a guiding principle for selecting anions to tailor threshold gas admission. This study not only overcomes the kinetic limitations related to gas admission in the "molecular trapdoor" zeolites but also underscores the potential of developing MOFs as molecular trapdoor adsorbents, providing valuable insights for designing ionic MOFs tailored to diverse gas separation applications.

Osa-miR1320 targets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling.

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.

A comprehensive investigation of the regulatory roles of OsERF096, an AP2/ERF transcription factor, in rice cold stress response.

KEY MESSAGE: OsERF096 negatively regulates rice cold tolerance and mediates IAA biosynthesis and signaling under cold stress. The APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors play important roles in regulating plant tolerance to abiotic stress. OsERF096 was previously identified as a direct target of miR1320, and was suggested to negatively regulate rice cold tolerance. In this study, we performed RNA-sequencing and targeted metabolomics assays to reveal the regulatory roles of OsERF096 in cold stress response. GO and KEGG analysis of differentially expressed genes showed that the starch and sucrose metabolism, plant-pathogen interaction, and plant hormone signal transduction pathways were significantly enriched. Quantification analysis confirmed a significant difference in sugar contents among WT and OsERF096 transgenic lines under cold treatment. Targeted metabolomics analysis uncovered that IAA accumulation and signaling were modified by OsERF096 in response to cold stress. Expectedly, qRT-PCR assays confirmed significant OsIAAs and OsARFs expression changes in OsERF096 transgenic lines. Finally, we identified three targets of OsERF096 based on RNA-seq, qRT-PCR, and dual-LUC assays. In summary, these results revealed the multiple regulatory roles of OsERF096 in cold stress response.

A Mannose Receptor from Litopenaeus vannamei Involved in Innate Immunity by Pathogen Recognition and Inflammation Regulation.

Mannose receptor, as a member of the C-type lectin superfamily, is a non-canonical pattern recognition receptor that can internalize pathogen-associated ligands and activate intracellular signaling. Here, a mannose receptor gene, LvMR, was identified from the Pacific white shrimp Litopenaeus vannamei. LvMR encoded a signal peptide, a fibronectin type II (FN II) domain, and two carbohydrate-recognition domains (CRDs) with special EPS and FND motifs. LvMR transcripts were mainly detected in the hepatopancreas, and presented a time-dependent response after pathogen challenge. The recombinant LvMR (rLvMR) could bind to various PAMPs and agglutinate microorganisms in a Ca2+-dependent manner with strong binding ability to D-mannose and N-acetyl sugars. The knockdown of LvMR enhanced the expression of most NF-κB pathway genes, inflammation and redox genes, while it had no obvious effect on the transcription of most phagocytosis genes. Moreover, the knockdown of LvMR caused an increase in reactive oxygen species (ROS) content and inducible nitric oxide synthase (iNOS) activity in the hepatopancreas after Vibrio parahaemolyticus infection. All these results indicate that LvMR might perform as a PRR in immune recognition and a negative regulator of inflammation during bacterial infection.

Genome-Wide Identification and Expression Analysis of UBiA Family Genes Associated with Abiotic Stress in Sunflowers (Helianthus annuus L.).

The UBiA genes encode a large class of isopentenyltransferases, which are involved in the synthesis of secondary metabolites such as chlorophyll and vitamin E. They performed important functions in the whole plant's growth and development. Current studies on UBiA genes were not comprehensive enough, especially for sunflower UBiA genes. In this study, 10 HaUBiAs were identified by domain analysis these HaUBiAs had five major conserved domains and were unevenly distributed on six chromosomes. By constructing phylogenetic trees, 119 UBiA genes were found in 12 species with different evolutionary levels and divided into five major groups, which contained seven conserved motifs and eight UBiA subsuper family domains. Tissue expression analysis showed that HaUBiAs were highly expressed in the roots, leaves, and seeds. By using promoter analysis, the cis-elements of UBiA genes were mainly in hormone signaling and stress responses. The qRT-PCR results showed that HaUBiA1 and HaUBiA5 responded strongly to abiotic stresses. Under ABA and MeJA treatments, HaUBiA1 significantly upregulated, while HaUBiA5 significantly decreased. Under cold stress, the expression of UBiA1 was significantly upregulated in the roots and stems, while UBiA5 expression was increased only in the leaves. Under anaerobic induction, UBiA1 and UBiA5 were both upregulated in the roots, stems and leaves. In summary, this study systematically classified the UBiA family and identified two abiotic stress candidate genes in the sunflower. It expands the understanding of the UBiA family and provides a theoretical basis for future abiotic stress studies in sunflowers.

Wild soybean SNARE proteins BET1s mediate the subcellular localization of the cytoplasmic receptor-like kinases CRCK1s to modulate salt stress responses.

Plants have evolved numerous receptor-like kinases (RLKs) that modulate environmental stress responses. However, little is known regarding soybean (Glycine max) RLKs. We have previously identified that Glycine soja Ca2+ /CAM-binding RLK (GsCBRLK) is involved in salt tolerance. Here, we report that soluble NSF attachment protein receptor proteins BET1s mediate subcellular localization of calmodulin-binding receptor-like cytoplasmic kinases CRCK1s to modulate salt stress responses. Direct interaction between GsCBRLK and GsBET11a was initially identified via yeast two-hybrid and bimolecular fluorescence complementation assays. Further analysis demonstrated conserved interaction between BET1s and CRCK1s. GsCBRLK interacted with all BET1 proteins in wild soybean (Glycine soja) and Arabidopsis, and GsBET11a strongly associated with GsCRCK1a-1d, but slightly with AtCRCK1. In addition, GsBET11a interacted with GsCBRLK via its C-terminal transmembrane domain (TMD), where the entire TMD, not the sequence, was critical for the interaction. Moreover, the N-terminal variable domain (VD) of GsCBRLK was responsible for interacting with GsBET11a, and the intensity of interaction between GsCBRLK/AtCRCK1 and GsBET11a was dependent on VD. Furthermore, GsBET11a was able to mediate the GsCBRLK subcellular localization via direct interaction with VD. Additionally, knockout of AtBET11 or AtBET12 individually did not alter GsCBRLK localization, while GsBET11a expression caused partial internalization of GsCBRLK from the plasma membrane (PM). We further suggest the necessity of GsCBRLK VD for its PM localization via N-terminal truncation assays. Finally, GsBET11a was shown to confer enhanced salt stress tolerance when overexpressed in Arabidopsis and soybean. These results revealed the conserved and direct interaction between BET1s and CRCK1s, and suggested their involvement in salt stress responses.

Influence of the thermodynamic properties of the Internal Occulter inside the coronagraph on coronal observations.

To detect good quality coronal spectra images, the continuous optimization of stray light suppression techniques for coronagraphs is required. The internal occulter (IO) serves as the main tool for the Internally Occulted Coronagraph to suppress the direct light from the photosphere layer, and thermal stress displacements with thermodynamic properties will overcover the information of the internal corona. In this paper, a reflective distribution function model is established according to Kirchhoff's principle which is based on a ground-based Lyot coronagraph, the aperture is 200 mm, detection wavelength is 637.4 nm (Fe X) and the work field range is ±1.05-2.0 RS (RS is the solar radius), thus the absorption rate is inverted. The irradiance at different positions received by the ground is simulated, and then the temperature change of the occulter during the time of the strongest radiation is calculated. The thermal stress displacement change of the two materials was analyzed by the finite element method. Comparison of the experiment shows that the displacement variation of the conical bottom plane results in losing 0.34% RS corona information for the 2a12-t6 aluminum alloy, and losing 0.11% RS coronal information for oxygen-free copper. This way provides a new idea for the thermodynamic modeling of the IO and the direct light suppression technology in the coronagraph.

Structure, Function, and Applications of Soybean Calcium Transporters.

Glycine max is a calcium-loving crop. The external application of calcium fertilizer is beneficial to the increase of soybean yield. Indeed, calcium is a vital nutrient in plant growth and development. As a core metal ion in signaling transduction, calcium content is maintained in dynamic balance under normal circumstances. Now, eight transporters were found to control the uptake and efflux of calcium. Though these calcium transporters have been identified through genome-wide analysis, only a few of them were functionally verified. Therefore, in this study, we summarized the current knowledge of soybean calcium transporters in structural features, expression characteristics, roles in stress response, and prospects. The above results will be helpful in understanding the function of cellular calcium transport and provide a theoretical basis for elevating soybean yield.

Genome-Wide Identification of AP2/ERF Transcription Factor Family and Functional Analysis of DcAP2/ERF#96 Associated with Abiotic Stress in Dendrobium catenatum.

APETALA2/Ethylene Responsive Factor (AP2/ERF) family plays important roles in reproductive development, stress responses and hormone responses in plants. However, AP2/ERF family has not been systematically studied in Dendrobium catenatum. In this study, 120 AP2/ERF family members were identified for the first time in D. catenatum, which were divided into four groups (AP2, RAV, ERF and DREB subfamily) according to phylogenetic analysis. Gene structures and conserved motif analysis showed that each DcAP2/ERF family gene contained at least one AP2 domain, and the distribution of motifs varied among subfamilies. Cis-element analysis indicated that DcAP2/ERF genes contained abundant cis-elements related to hormone signaling and stress response. To further identify potential genes involved in drought stress, 12 genes were selected to detect their expression under drought treatment through qRT-PCR analysis and DcAP2/ERF#96, a nuclear localized ethylene-responsive transcription factor, showed a strong response to PEG treatment. Overexpression of DcAP2/ERF#96 in Arabidopsis showed sensitivity to ABA. Molecular, biochemical and genetic assays indicated that DcAP2ERF#96 interacts with DREB2A and directly inhibits the expression of P5CS1 in response to the ABA signal. Taken together, our study provided a molecular basis for the intensive study of DcAP2/ERF genes and revealed the biological function of DcAP2ERF#96 involved in the ABA signal.

Adsorption and visible-light photocatalytic degradation of organic pollutants by functionalized biochar: Role of iodine doping and reactive species.

Here we developed the functionalized biochar as low-cost and heavy metal-free photocatalysts via a facile iodine doping method, which exhibit efficient adsorption and visible-light-driven photocatalytic degradation of representative organic pollutants, phenol and tetracycline. On one hand, iodine doping elevates the adsorption via creating extra pores, e.g., the adsorbed amounts of phenol by iodine-doped WSP and OSR biochar are increased by 161.8% and 146.3%, respectively, which in turn facilitates the photocatalytic oxidation of the adsorbed pollutants. On the other hand, iodine doping leads to the strong photo-induced excitation and remarkably reduced charge carrier transfer resistance, boosting the photocatalytic activity of iodine-doped biochar by more than 20 orders towards organic pollutants (e.g., phenol) degradation. The systematic analysis of reactive species reveals the active roles of O2-, H2O2, 1O2, OH, electrons, and holes in photocatalytic process and identifies O2- to be the major contributor. This work affords a facile approach to generating porous and visible-light-driven photocatalyst from biomass for efficient adsorbing and degrading organic pollutants, opening up an avenue to turn biowaste into biomaterials for sustainable environmental remediation.

A Lymphoid Organ Specific Anti-Lipopolysaccharide Factor from Litopenaeus vannamei Exhibits Strong Antimicrobial Activities.

Different shrimp species are known to possess apparent distinct resistance to different pathogens in aquaculture. However, the molecular mechanism underlying this finding still remains unknown. One kind of important antimicrobial peptides, anti-lipopolysaccharide factors (ALF), exhibit broad-spectrum antimicrobial activities. Here, we reported a newly identified ALF from the shrimp Litopenaeus vannamei and compared the immune function with its counterpart in the shrimp Fenneropenaeus chinensis. The ALF, designated as LvALF8, was specifically expressed in the lymphoid organ of L. vannamei. The expression level of LvALF8 was apparently changed after white spot syndrome virus (WSSV) or Vibrio parahaemolyticus challenges. The synthetic LBD peptide of LvALF8 (LvALF8-LBD) showed strong antibacterial activities against most tested Gram-negative and Gram-positive bacteria. LvALF8-LBD could also inhibit the in vivo propagation of WSSV similar as FcALF8-LBD, the LBD of LvALF8 counterpart in F. chinensis. However, LvALF8-LBD and FcALF8-LBD exhibited apparently different antibacterial activity against V. parahaemolyticus, the main pathogen causing acute hepatopancreatic necrosis disease (AHPND) of affected shrimp. A structural analysis showed that the positive net charge and amphipathicity characteristics of LvALF8-LBD peptide were speculated as two important components for its enhanced antimicrobial activity compared to those of FcALF8-LBD. These new findings may not only provide some evidence to explain the distinct disease resistance among different shrimp species, but also lay out new research ground for the testing and development of LBD-originated antimicrobial peptides to control of shrimp diseases.

Genome-Wide Identification, Characterization and Expression Analysis of Soybean CHYR Gene Family.

The CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) proteins have been functionally characterized in iron regulation and stress response in Arabidopsis, rice and Populus. However, their roles in soybean have not yet been systematically investigated. Here, in this study, 16 GmCHYR genes with conserved Zinc_ribbon, CHY zinc finger and Ring finger domains were obtained and divided into three groups. Moreover, additional 2-3 hemerythrin domains could be found in the N terminus of Group III. Phylogenetic and homology analysis of CHYRs in green plants indicated that three groups might originate from different ancestors. Expectedly, GmCHYR genes shared similar conserved domains/motifs distribution within the same group. Gene expression analysis uncovered their special expression patterns in different soybean tissues/organs and under various abiotic stresses. Group I and II members were mainly involved in salt and alkaline stresses. The expression of Group III members was induced/repressed by dehydration, salt and alkaline stresses, indicating their diverse roles in response to abiotic stress. In conclusion, our work will benefit for further revealing the biological roles of GmCHYRs.

The sea cucumber genome provides insights into morphological evolution and visceral regeneration.

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

Performance Recovery in Degraded Carbon-Based Electrodes for Capacitive Deionization.

Limitations of capacitive deionization (CDI) and future commercialization efforts are intrinsically bound to electrode stability. In this work, thermal treatments are explored to understand their ability to regenerate aged CDI electrodes. We demonstrate that a relatively low thermal treatment temperature of ∼500 °C can sufficiently recover the lost salt adsorption capacity of degraded electrodes. Furthermore, a systematic study of electrode replacement clarifies that the desalination ability loss and regeneration for a CDI cell are isolated to the aged anode, as expected. Characterizations of surface functionalities support that the acidic oxygen-containing functional groups formed in situ during cycling undergo thermal decomposition during treatment. The modified Donnan model quantitatively confirms that the surface charges originate from the formation/decomposition of functional groups. Accordingly, the lost pore volume and the increased resistance are recovered during thermal treatments, while the surface morphologies and pore structure of the electrodes are well-preserved. Therefore, thermal treatment can be applied practically to extend the lifetime of aged electrodes. This study also offers insights into strategies for minimizing electrode degradation or in situ regeneration such that the technology gains momentum for future commercialization.

Photocatalytic Bacterial Inactivation by a Rape Pollen-MoS2 Biohybrid Catalyst: Synergetic Effects and Inactivation Mechanisms.

A novel and efficient 3D biohybrid photocatalyst, defective MoS2 nanosheets encapsulated carbonized rape pollen, was fabricated and applied to water disinfection. The rape pollen-MoS2 (PM) biohybrid showed excellent dispersibility, high stability, and efficient charge-carrier separation and migration ability, resulting in the highly enhanced photocatalytic inactivation performance toward various waterborne bacteria under different light sources. The inactivation mechanisms were systematically investigated. Reactive species (RSs), including electrons, holes, and reactive oxygen species (•O2- and •OH), played major roles in inactivating bacteria. The antioxidant system of bacteria exhibited a self-protection capacity by eliminating the photogenerated RSs from PM biohybrid at the early stage of inactivation. With the accumulation of RSs, the cell membrane and membrane-associated functions were destroyed, as suggested by the collapse of cell envelope and subsequent loss of cell respiration and ATP synthesis capacity. The microscopic images further confirmed the destruction of the bacterial membrane. After losing the membrane barrier, the oxidation of cytoplasmic proteins and lipids caused by invaded RSs occurred readily. Finally, the leakage of DNA and RNA announced the irreversible death of bacteria. These results indicated that the bacterial inactivation began with the membrane rupture, followed by the oxidation and leakage of intracellular substances. This work not only provided a new insight into the combination of semiconductors with earth-abundant biomaterials for fabricating high-performance photocatalysts, but also revealed the underlying mechanisms of photocatalytic bacterial inactivation in depth.

Correction to: A Glycine soja group S2 bZIP transcription factor GsbZIP67 conferred bicarbonate alkaline tolerance in Medicago sativa.

Following publication of the original article [1], the author reported that their given name was misspelled.

Dynamic extraction coupled on-line to liquid chromatography with a parallel sampling interface-a proof of concept for monitoring extraction kinetics.

On-line hyphenation of extraction with chromatography has been explored in several different types of combinations. However, monitoring the complete process of a dynamic, continuous-flow extraction is not possible with any hyphenated system reported so far. The current work demonstrates that this challenging task can be effectively fulfilled by using a parallel sampling interface, which mimics the concept of comprehensive two-dimensional chromatography. In this study, pressurised hot water extraction was coupled on-line with ultra-high-performance liquid chromatography. The set-up was utilised in a kinetic study of dynamic pressurised hot water extraction of curcuminoids from turmeric powder. Compound-specific extraction curves were obtained, which clearly indicated the rate-limiting factors of the extraction processes under different conditions. Additionally, thermal degradation of curcumin during the extraction could also be demonstrated in some of the extractions.

Signal enhancement in supercritical fluid chromatography-diode-array detection with multiple injection.

To circumvent the detrimental effects of large-volume injection with fixed-loop injector in modern supercritical fluid chromatography, the feasibility of performing multiple injection was investigated. By accumulating analytes from a certain number of continual small-volume injections, compounds can be concentrated on the column head, and this leads to signal enhancement compared with a single injection. The signal to noise enhancement of different compounds appeared to be associated with their retention on different stationary phases and with type of sample diluent. The diethylamine column gave the best signal to noise enhancement when acetonitrile was used as sample diluent and the 2-picolylamine column showed the best overall performance with water as the sample diluent. The advantage of multiple injection over one-time large-volume injection was proven with sulfanilamide, with both acetonitrile and water as sample diluents. The multiple injection approach exhibited comparable within- and between-day precision of retention time and peak area with those of single injections. The potential of the multiple injection approach was demonstrated in the analysis of sulfanilamide-spiked honey extract and diclofenac-spiked ground water sample. The limitations of this approach were also discussed.

A Glycine soja group S2 bZIP transcription factor GsbZIP67 conferred bicarbonate alkaline tolerance in Medicago sativa.

BACKGROUND: Even though bicarbonate alkaline stress is a serious threat to crop growth and yields, it attracts much fewer researches than high salinity stress. The basic leucine zipper (bZIP) transcription factors have been well demonstrated to function in diverse abiotic stresses; however, their biological role in alkaline tolerance still remains elusive. In this study, we functionally characterized a bZIP gene from Glycine soja GsbZIP67 in bicarbonate alkaline stress responses. RESULTS: GsbZIP67 was initially identified as a putative bicarbonate responsive gene, on the basis of previous RNA-seq data of 50 mM NaHCO3-treated Glycine soja roots. GsbZIP67 protein possessed a conserved bZIP domain, and belonged to the group S2 bZIP, which is yet less well-studied. Our studies showed that GsbZIP67 targeted to nucleus in Arabidopsis protoplasts, and displayed transcriptional activation activity in yeast cells. The quantitative real-time PCR analyses unraveled the bicarbonate stress responsive expression and tissue specific expression of GsbZIP67 in wild soybean. Further phenotypic analysis illustrated that GsbZIP67 overexpression in alfalfa promoted plant growth under bicarbonate alkaline stress, as evidenced by longer roots and shoots. Furthermore, GsbZIP67 overexpression also modified the physiological indices of transgenic alfalfa under bicarbonate alkaline stress. In addition, the expression levels of several stress responsive genes were also augmented by GsbZIP67 overexpression. CONCLUSIONS: Collectively, in this study, we demonstrated that GsbZIP67 acted as a positive regulator of plant tolerance to bicarbonate alkaline stress. These results provide direct genetic evidence of group S2 bZIPs in bicarbonate alkaline stress, and will facilitate further studies concerning the cis-elements and/or downstream genes targeted by GsbZIP67 in stress responses.