Bimolecular Fluorescence Complementation Assays Research Articles

The SEPALLATA-like gene HrSEP1 in Hippophae rhamnoides regulates flower development by interacting with other MADS-box subfamily genes

MADS-box genes are classified into five categories: ABCDE, including SEP1, SEP2, SEP3, SEP4, and other homologous genes, which play important roles in floral organ development. In this study, the cDNA sequence of the HrSEP1 gene was cloned by RT-PCR and confirmed that this gene belongs to the MADS-box gene family. In addition, subcellular localization experiments showed that the HrSEP1 protein was localized in the nucleus. We verified the interaction of HrSEP1 with HrSOC1, HrSVP, and HrAP1 using yeast two-hybrid and bimolecular fluorescence complementation assays. These genes jointly regulate the growth and development of floral organs. We also found a strong synergy between HrSEP1 and AP1 genes in sepals, petals, and stamens by transgenic methods and fluorescence quantitative PCR, suggesting that HrSEP1 and AP1 may co-regulate the development of these structures. In conclusion, the expression of HrSEP1 has a certain effect on the development of floral organs, and these findings lay the foundation for further research on the biological functions of MADS transcription factors in Hippophae rhamnoides.

Visualizing Tomato Spotted Wilt Virus Protein Localization: Cross-Kingdom Comparisons of Protein-Protein Interactions.

Tomato spotted wilt virus (TSWV) is an orthotospovirus that infects both plants and insect vectors, and understanding its protein localization and interactions is crucial for unraveling the infection cycle and host-virus interactions. We investigated and compared the localization of TSWV proteins. The localization between plant and insect cells was overall consistent, indicating a similar mechanism is utilized by the virus in both types of cells. However, a change in localization over time was associated with the viral proteins that did not contain signal peptides and transmembrane domains such as N, NSs, and NSm, which only occurred in the plant cells and not in the insect cells. We also tested the localization of the proteins during an active plant infection using free red fluorescent protein (RFP) as a marker to highlight the nucleus and cytoplasm. Voids in the cytoplasm were shown only during infection, and N, NSs, NSm, and to a lesser extent GN and GC were surrounding these areas, suggesting it may be a site of replication or morphogenesis. Furthermore, we tested the interactions of viral proteins using both bimolecular fluorescence complementation (BiFC) and membrane-based yeast two-hybrid (MbY2H) assays. These revealed self-interactions of NSm, N, GN, GC, and NSs. We also identified interactions between different TSWV proteins, indicating their possible roles, such as between NSs and GC and N and GC, which may be necessary during the replication and assembly processes, respectively. This research expands our knowledge of TSWV infection and elaborates on the intricate relationships between viral proteins, cellular dynamics, and host responses. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The synergistic roles of MsRCI2B and MsRCI2E in the regulation of ion balance and ROS homeostasis in alfalfa under salt stress.

Under salt stress, plasma membrane proteins regulate ion homeostasis and the balance between reactive oxygen species (ROS). In this study, we investigated the functions of two small membrane proteins-MsRCI2B (tailless) and MsRCI2E (tailed)-encoded by the RCI2 (Rare Cold Inducible 2) gene family in Medicago sativa (alfalfa). We identified the distinct subcellular localization and expression patterns of these proteins under salt stress. Using yeast two-hybrid (Y2H), GST pull-down, and bimolecular fluorescence complementation (BiFC) assays, we confirmed the physical interactions between MsRCI2B and MsRCI2E. Transgenic alfalfa lines overexpressing MsRCI2(OE#RCI2) and co-expressing both MsRCI2B and MsRCI2E (OE#RCI2E-2B) were developed to explore their roles in salt tolerance. Interestingly, the C-terminal tail of MsRCI2E negatively affects salt tolerance; however, its interaction with MsRCI2B mitigates this adverse effect. To further understand the regulatory mechanisms, we screened for plasma membrane proteins (PMPs) that interact with MsRCI2B or MsRCI2E using a DUALmembrane yeast two-hybrid system. MsCaM1 interacts with MsRCI2B, whereas MsPIP1;4 and MsHVP1 specifically interact with MsRCI2E. Notably, the MsRCI2E-PIP1;4 interaction influenced the intracellular trafficking of PIP1;4, reducing its presence on the plasma membrane and thereby limiting the export of H2O2, which helps maintain ROS homeostasis. Additionally, the interaction between MsRCI2E and HVP1 promoted ion homeostasis by decreasing Na+ influx under salt stress. Overall, our study provides new insights into the molecular mechanisms through which MsRCI2B and MsRCI2E coordinate the ion and ROS balance under salt stress and offering promising strategies for enhancing crop tolerance to salinity.

TaSnRK3.23B, a CBL-interacting protein kinase of wheat, confers drought stress tolerance by promoting ROS scavenging in Arabidopsis

BackgroundSucrose non-fermenting-1-related protein kinases (SnRKs) have been implicated in plant growth and stress responses. Although SnRK3.23 is known to be involved in drought stress, the underlying mechanism of resistance differs between Arabidopsis and rice, and little is known about its function in wheat.ResultsIn the current work, TaSnRK3.23B was detected on the cell membrane and in the nucleus. TaSnRK3.23B overexpression in Arabidopsis promoted reactive oxygen species (ROS) scavenging via the accumulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) and then conferred significant tolerance to drought. The prediction analysis, yeast two-hybrid, and bimolecular fluorescence complementation (BiFC) assays revealed that TaSnRK3.23B interacted with TaCBL2B and TaCBL6B, which are calcineurin B-like (CBL) proteins. The predicted model demonstrated that TaSnRK3 subfamily proteins participate in Ca2+ signaling mediated by TaCBL2B/TaCBL6B and subsequently provide drought stress tolerance by promoting ROS scavenging in wheat.ConclusionsAltogether, the obtained findings contribute to a better understanding of the functions of SnRK3.23 in wheat and offer genetic suggestions for improving drought resistance of wheat.

The petunia heavy metal P-type ATPase PhHMA5II1 interacts with copper chaperons and regulate Cu detoxification.

An endoplasmic reticulum-localized Cu transporter, PhHMA5II1, interacts with copper chaperones and plays an important role in Cu detoxification in petunia. Copper (Cu) is an essential element for plant growth but toxic when present in excess. In this study we present the functional characterization of a petunia (Petunia hybrida) P1B-type heavy-metal ATPases (HMAs), PhHMA5II1. Heterologous expression of PhHMA5II1 in yeast (Saccharomyces cerevisiae) showed Cu transport activity. The expression of PhHMA5II1 in roots and shoots was unaffected by excess Cu. CRISPR/Cas9-edited mutant lines and PhHMA5II1 overexpressing transgenic plants were generated to investigate the functions of PhHMA5II1 in petunia. The PhHMA5II1 knockout mutant was hypersensitive to excess Cu and accumulated more Cu in roots compared to wild-type petunia. Overexpression of PhHMA5II1 enhanced Cu tolerance and reduced Cu accumulation in roots. Furthermore, PhHMA5II1 localized in endoplasmic reticulum, and the localization was unaffected by excess Cu. Yeast two-hybrid experiments and bimolecular fluorescence complementation assays demonstrate that PhHMA5II1 interact with petunia copper chaperons, PhATX1 and PhCCH. Finally, RNA-sequencing revealed that knockout PhHMA5II1 affected the expression of genes involved in cell-wall organization, copper ion homeostasis, and response to oxidative stress. Taken together, PhHMA5II1 plays an important role in Cu detoxification in petunia.

Sugarcane Pan-Transcriptome Identifying a Master Gene ScHCT Regulating Lignin and Sugar Traits.

Sugarcane has the most complex polyploid genome in the world, and sugar-related traits are one of the most important aims in sugarcane breeding. It is essential to construct a representative pan-transcriptome that contains all transcripts of a species for studies on genetic diversity, population expression, and omics analyses in sugarcane. In this study, we constructed the first comprehensive pan-transcriptome for sugarcane, and 8434 highly reliable open reading frames were found, which were not aligned with any published sugarcane genome. The core and dispensable gene clusters, as well as high- and low-expression gene clusters of the pan-transcriptome, were identified and analyzed. The integration of two sugar content differential transcriptome data revealed nine key candidate genes, including the ScHCT gene, encoding a crucial enzyme for lignin synthesis. Furthermore, the function of the ScHCT gene was validated inArabidopsis, which was negatively correlated with sugar content and positively correlated with lignin content. The interaction protein of ScHCT, ScABH, was screened via a yeast two-hybrid assay and further validated by point-to-point Y2H and bimolecular fluorescence complementation assays. The phenotype of the Arabidopsis abh mutant line revealed that loss of function of ABH resulted in a decrease of sucrose content in stem tissue. This study provides important reference information and genetic resources for sugarcane research and varietal improvement.

TaFAR9 and TaFAR10 synergistically regulate fertility conversion of photo-thermo-sensitive genic male sterility lines in wheat by modulating ROS homeostasis

Photo-thermo-sensitive genic male sterility (PTGMS), which exhibits varying fertility levels under different environmental conditions, is a crucial method for heterosis utilization in wheat. However, the mechanisms underlying fertility conversion remain unclear. In the study, three BS type PTGMS lines were analyzed to study fertility conversion characteristics. The results indicated that the fertility conversion occurred during meiosis and was accompanied by an increase in reactive oxygen species (ROS) under a sterile environment. TaFAR9 encoding a novel fatty acyl CoA reductase was identified using transcriptome sequencing. Expression analysis suggested that TaFAR9 was localized in the endoplasmic reticulum (ER), with high expression levels in anthers. Furthermore, the down-regulation of TaFAR9 expression displayed characteristics of male sterility, accompanied by the accumulation of ROS. Cytological analysis revealed abnormal development in the anther and pollen walls of TaFAR9-silenced lines. Additionally, TaFAR9 and TaFAR10 were confirmed to physically interact using molecular docking simulation, yeast two-hybrid, luciferase complementation, and bimolecular fluorescence complementation assays. The reduced expression of TaFAR10 also exhibited male sterility and ROS burst. Moreover, the co-silencing of TaFAR9 and TaFAR10 produced sterility phenotypes that were similar to those observed when silencing TaFAR9 or TaFAR10 individually. Transcriptome analysis suggested that the ROS burst in BSMV: TaFAR9/10 anthers can result in cellular metabolic disorders. These findings indicate that TaFAR9 and TaFAR10 may form heterodimers that synergistically regulate fertility conversion in PTGMS lines by modulating ROS metabolism. And this study offers a fresh insight into the regulatory processes involved in fertility conversion in PTGMS lines.

Identification and characterization of interacting proteins of transcription factor DpWRI1-like related to lipid biosynthesis from microalga Dunaliella parva.

Our previous study found that Dunaliella parva WRINKLED1-like (DpWRI1-like) was a key regulatory factor of lipid biosynthesis in D. parva. DpWRI1-like gene and target genes of DpWRI1-like have been obtained in our previous study, but the interacting proteins of DpWRI1-like are unclear now, which has limited a deep understanding of the function of DpWRI1-like. Yeast two-hybrid was widely used to identify protein-protein interaction. In this study, the interacting proteins of DpWRI1-like were obtained using yeast two-hybrid technique to further realize the role of DpWRI1-like. Three important interacting proteins have the following predicted activities: acyl-CoA-binding domain-containing protein 6 (interacting protein 1, ACBD6), duplicated carbonic anhydrase (interacting protein 2, DCA) and DNA-binding transcription factor (interacting protein 3, TF). Bimolecular fluorescence complementation assay further validated the interaction between DpWRI1-like and interacting proteins ACBD6 and DCA. The further bioinformatics analyses of interacting proteins were conducted. Protein-protein docking indicated the strong affinity between DpWRI1-like and three interacting proteins. Since interacting proteins have been found to be related to lipid biosynthesis in other organisms, this study contributes to a deeper understanding of the role of DpWRI1-like in lipid synthesis. In conclusion, this study firstly reported three interacting proteins (ACBD6, DCA and TF) of DpWRI1-like related to lipid biosynthesis, and conducted their bioinformatics analyses, which would be conducive to a deep understanding of the function of DpWRI1-like in lipid biosynthesis.

Design of Small Non-Peptidic Ligands That Alter Heteromerization between Cannabinoid CB1 and Serotonin 5HT2A Receptors.

Activation of cannabinoid CB1 receptors (CB1R) by agonists induces analgesia but also induces cognitive impairment through the heteromer formed between CB1R and the serotonin 5HT2A receptor (5HT2AR). This side effect poses a serious drawback in the therapeutic use of cannabis for pain alleviation. Peptides designed from the transmembrane helices of CB1R, which are predicted to bind 5HT2AR and alter the stability of the CB1R-5HT2AR heteromer, have been shown to avert CB1R agonist-induced cognitive impairment while preserving analgesia. Using these peptides as templates, we have now designed nonpeptidic small molecules that prevent CB1R-5HT2AR heteromerization in bimolecular fluorescence complementation assays and the heteromerization-dependent allosteric modulations in cell signaling experiments. These results provide proof-of-principle for the design of optimized ligand-based disruptors of the CB1R-5HT2AR heteromer, opening new perspectives for in vivo studies.

The role of sugar transporter BrSWEET11 in promoting plant early flowering and preliminary exploration of its molecular mechanism.

BrSWEET11 accelerated Arabidopsis thaliana flowering, while silencing Brsweet11 in Brassica rapa delayed flowering relative to controls. BrSWEET11 is involved in sucrose transport after being induced by long-day conditions. SWEETs (Sugars Will Eventually Be Exported Transporters) are sugar outflow transporters that may participate in the regulation of plant flowering. In this study, the open reading frame of Brassica rapa ssp. pekinensis SWEET11 (BrSWEET11) was cloned and found to be 858bp in length and encode 285 amino acids, which is typical of SWEET family proteins. The BrSWEET11 gene was strongly expressed in reproductive growth organs, particularly flowers, according to tissue expression analyses and GUS histochemical staining. BrSWEET11 promotes early flowering in Arabidopsis thaliana by 3-4days, whereas Brsweet11 silencing in Brassica rapa delays flowering by 8-12days relative to controls. BrSWEET11 promoted early flowering in A. thaliana, and compared with that in control plants, flowering was delayed in Brsweet11-silenced Brassica rapa. Transcriptome analysis of BrSWEET11-overexpressing A. thaliana and wild-type (WT) plants was performed and the results showed that eight key flowering genes jointly regulated flowering time, which was also validated in the Brsweet11-silenced plants. In addition, through photoperiod treatments and sugar content measurements, it was found that the expression of BrSWEET11 is induced by long-day conditions and is involved in sucrose transport. Further investigation using yeast library screening, yeast two-hybrid, and bimolecular fluorescence complementation assay techniques revealed that the BrSWEET11 protein interacts with the sugar transporter 4a (BrSUT4a) protein. Therefore, BrSWEET11 was induced by long-day conditions, and may promote early flowering in Brassica rapa through sucrose transport. This study provides a theoretical basis for elucidating the molecular mechanism through which SWEET genes are involved in flowering time regulation in Brassica rapa.

OsPAD1, encoding a non-specific lipid transfer protein, is required for rice pollen aperture formation.

Plant lipid transfer proteins (LTPs) are distinguished by their capacity to facilitate lipid transport in vitro between membranes. This includes the transportation of lipid constituents from the tapetum to the microspore, thereby playing a pivotal role in the synthesis and construction of the pollen wall, encompassing the formation of the pollen aperture. However, our understanding of LTPs and their role in pollen aperture formation in rice remains limited. In this study, we have isolated and characterized a male sterile rice mutant named as pollen aperture defect 1 (Ospad1). When compared to the wild type, Ospad1 mutant plants exhibit pollen grain abortion due to the absence of the fibrillar-granular layer, ultimately leading to the leakage of contents from the malformed aperture. OsPAD1 encodes a non-specific LTP and is specifically expressed in the microspore during male development. Subsequently, in vitro lipid binding assays reveal that the recombinant OsPAD1 protein has the capability to bind to a broad spectrum of lipids. The malfunction of OsPAD1 results in disrupted lipid metabolism and compromised pollen aperture, ultimately leading to male sterility. Furthermore, yeast two-hybrid, bimolecular fluorescent complementation and pull-down assays all demonstrate that OsPAD1 can directly interact with OsINP1, an orthologue of a crucial aperture factor in Arabidopsis, together regulating rice aperture development. These findings offer new insights into the molecular mechanisms that underlie the function of LTPs in rice pollen aperture formation. This research holds potential implications not only for rice but also for other cereal crops.

SlUPA-like, a bHLH Transcription Factor in Tomato (Solanum lycopersicum), Serves as the Crosstalk of GA, JA and BR.

The bHLH (basic Helix-Loop-Helix) transcription factor serves as pivotal controller in plant growth and development. In a previous study, the overexpression of SlUPA-like in Solanum lycopersicum L. Ailsa Craig (AC++) altered the JA (Jasmonic acid) response and endogenous GA (Gibberellic acid) content. However, the detailed regulation mechanism was not fully explored. In the present research, we found that the overexpression of SlUPA-like influenced the accumulation of GA, JA and BR (Brassinolide). RNA-Seq data illustrated that the expression levels of genes related to these plant hormones were significantly affected. Additionally, the interaction of SlUPA-like with SlMYB21, SlMYC2 and SlDELLA was characterized by employing Y2H (Yeast Two-Hybrid) and BiFC (Bimolecular Fluorescence Complementation) assay. Furthermore, Dual-LUC (Dual-Luciferase) assay and EMSA (Electrophoretic Mobility Shift Assay) identified that SlUPA-like directly targeted the E-box motif in the promoter of SlGID2 and activated the transcription of SlGID2. These results shed light on the potential role of SlUPA-like in mediating crosstalk among multiple plant hormones and established a robust theoretical framework for further unraveling the functions of SlUPA-like transcription factors in the context of plant growth and hormone signal transduction.

Rice small protein OsS1Fa1 participates in stress responses as an inner nuclear membrane protein

ABSTRACT The rice small protein OsS1Fa1, a homolog of spinach S1Fa, plays a significant role in drought tolerance, attributed to its transmembrane domain. In this study, we aim to further elucidate the potential roles of OsS1Fa1 in cold and biotic stresses as an inner nuclear membrane protein. Fluorescence analysis confirmed the localization of OsS1Fa1 to the inner nuclear membrane. Utilizing the bimolecular fluorescence complementation (BiFC) and bacterial infiltration assays with OsS1Fa1 and the inner nuclear membrane protein OsSUN1 (Rice Sad1 and UNC84 (SUN) domain containing 1 (SUN1)), we observed fluorescence detection within the inner nuclear membrane, indicating a direct interaction and colocalization between OsS1Fa1 and OsSUN1. Expression analysis revealed that overexpression of OsS1Fa1 induced the expression of various genes associated with cold and defense responses, including COLD-REGULATED 15A (COR15A), PATHOGENESIS-RELATED PROTEIN 1 (PR1), and PLANT DEFENSIN 1.2 (PDF1.2). Our findings collectively indicate that OsS1Fa1 plays crucial roles in both abiotic and biotic stress tolerance as an inner nuclear membrane protein.

ATP-binding cassette G23 is required for Arabidopsis seed coat suberization.

Suberin is an extracellular hydrophobic polymer deposited in seed coats that acts as a barrier to regulate the movement of ions, water, and gases, and protects seeds against pathogens. However, the molecular mechanisms underlying suberin deposition in the seed coat remain unknown. In this study, the in planta role of ATP-binding cassette G23 (ABCG23) was investigated in the Arabidopsis seed coat. ABCG23 transcripts were predominantly expressed in the outer integument1 (oi1) of seed coats and the endodermal cells of roots. The fluorescence of the eYFP:ABCG23 construct was observed in the plasma membranes of the tobacco epidermis, seed coat oi1, and root endodermal cells. Seed coats of abcg23-1 and abcg23-2 mutants exhibited reduced autofluorescence under UV light and increased permeability to tetrazolium salts. Total suberin loads and major suberin components, C24 ω-hydroxy fatty acids and 1, ω-dicarboxylic acids were significantly decreased in the mutant seed coats. The ratio of seed germination and seedling establishment of abcg23-1 and abcg23-2 was significantly reduced compared to the WT under salt and osmotic stress conditions. The bimolecular fluorescence complementation assay showed homodimeric interactions of ABCG-2, -6, -20, and -23 and heterodimeric interactions between ABCG23 and ABCG-2, -6, -11, or -20. Our findings indicate that ABCG23 contributes to the transport of suberin monomers in the Arabidopsis seed coats.

LRR1 involved in the abscisic acid signaling pathway to regulate the early growth and development of Arabidopsis thaliana.

Living organisms possess the remarkable capacity to swiftly adapt to fluctuations in their environment. In the context of cell signal transduction, a significant challenge lies in ensuring the effective perception of external signals and the execution of appropriate responses. To investigate this phenomenon, a recent study utilized Arabidopsis thaliana as a model plant and induced stress by administering abscisic acid (ABA), a plant hormone, to elucidate the involvement of leucine-rich repeat receptor-like kinase1 (LRR1) in ABA signaling pathways. Homozygous T-DNA insertion alleles for LRR1 and KIN7 were isolated. Quantitative real-time PCR (qRT-PCR) was performed to confirm the expression of the LRR1 gene. Subcellular localization and beta-glucuronidase (GUS) tissue labeling techniques were utilized to determine the expression pattern of the LRR1 gene in cells and tissues. Yeast two-hybrid complementation, bimolecular fluorescence complementation assay, and GST pull-down assays were conducted to validate the interaction of LRR1 proteins. Phenotypic analyses revealed that lrr1 and kin7 mutants are less sensitive to the inhibitory effects of ABA on germination and cotyledon greening that is seen in WT. Mutants LRR1 and kinase 7 (KIN7) exhibited resistance to ABA and displayed normal growth patterns under control conditions. The double mutant lrr1kin7 showed reduced responsiveness to ABA. Conversely, overexpression lines LRR1ox2 and LRR1ox10 demonstrated heightened sensitivity to ABA, resulting in severe growth reduction. qRT-PCR assay indicated that exogenous application of ABA led to significant down-regulation of ABI3, ABI4, and ABI5 transcription factors in LRR1 material compared to wild-type WT material. An investigation was conducted to determine the expression pattern and transcriptional level of LRR1 in Arabidopsis. The results revealed ubiquitous expression of LRR1 across all developmental stages and tissue tested. Subcellular localization assays confirmed the presence of LRR1 on the plasma membrane of cells. Furthermore, BiFC assay, yeast two-hybrid complementation, and GST pull-down assays demonstrated an interaction between LRR1 and PYL6 in vitro. These findings provide substantial insights into the involvement of LRR1 in the ABA signaling pathway while regulating seed germination and cotyledon greening during early development in Arabidopsis. This study significantly advances our understanding regarding the correlation between LRR1 and ABA signaling pathways with potential applications for enhancing crop stress resistance.

A C2H2-type zinc finger protein TaZFP8-5B negatively regulates disease resistance

BackgroundZinc finger proteins (ZFPs) are important regulators in abiotic and biotic stress tolerance in plants. However, the role of the ZFPs in wheat responding to pathogen infection is poorly understood.ResultsIn this study, we found TaZFP8-5B was down-regulated by Puccinia striiformis f. sp. tritici (Pst) infection. TaZFP8-5B possesses a single C2H2-type zinc finger domain with a plant-specific QALGGH motif, and an EAR motif (LxLxL) at the C-terminus. The EAR motif represses the trans-activation ability of TaZFP8-5B. Knocking down the expression of TaZFP8 by virus-induced gene silencing increased wheat resistance to Pst, whereas TaZFP8-5B-overexpressing reduced wheat resistance to stripe rust and rice resistance to Magnaporthe oryzae, suggesting that TaZFP8-5B plays a negative role in the modulation of plant immunity. Using bimolecular fluorescence complementation, split-luciferase, and yeast two-hybrid assays, we showed that TaZFP8-5B interacted with a wheat calmodulin-like protein TaCML21. Knock-down of TaCML21 reduced wheat resistance to Pst.ConclusionsThis study characterized the function of TaZFP8-5B and its interacting protein TaCML21. Our findings provide a new perspective on a regulatory module made up of TaCML21-TaZFP8-5B in plant immunity.

Wheat Tae-MIR1118 Constitutes a Functional Module With Calmodulin TaCaM2-1 and MYB Member TaMYB44 to Modulate Plant Low-N Stress Response.

Distinct target genes are modulated by microRNA members and affect various biological processes associated with abiotic stress responses in plants. In this study, we characterized a functional module comprising miRNA/target and a downstream MYB transcription factor partner, Tae-MIR1118/TaCaM2/TaMYB44, in Triticum aestivum to mediate the plant low-nitrogen (N) stress response. Dual luciferase (LUC) assay and expression analysis indicated that TaCaM2 is regulated by Tae-MIR1118 through a posttranscriptional cleavage mechanism. Reporter LUC activity in N. benthamiana leaves co-transformed with effector CaMV35S::Tae-MIR1118 and reporter TaCaM2::LUC was significantly reduced, and the transcripts of Tae-MIR1118 and TaCaM2 in tissues exhibited converse expression patterns under varying N levels. Specifically, the transcripts of Tae-MIR1118 decreased, whereas those of TaCaM2 increased under low-N stress in a temporal-dependent manner. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays indicated that TaCaM2 interacted with the MYB transcription factor TaMYB44. Transgene analysis revealed the negative roles of Tae-MIR1118 and the positive functions of TaCaM2 and TaMYB44 in regulating plants for low-N stress adaptation by modulating glutamine synthetase activity, N uptake capacity, and root morphology. Yeast one-hybrid, transcriptional activation, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-PCR) assays indicated that TaMYB44 could bind to the promoters of genes TaGS2.2, TaNRT2.1, and TaPIN4 and induce transcription of these stress-defensive genes. Knockdown of these three genes reduced GS activity, N accumulation, and root growth traits in plants subjected to N starvation. The yield in the wheat variety panel was highly correlated with the transcripts of Tae-MIR1118, TaCaM2, and TaMYB44 in plants cultured under N-deprived field conditions. A major haplotype of Tae-MIR1118, TaMIR1118-Hap1, enhanced the low-N stress tolerance of plants. Our findings indicate that the Tae-MIR1118/TaCaM2/TaMYB44 pathway primarily affects the low-N response of plants by modulating associated physiological processes.

Orf116b Induces Pollen Abortion in a Novel Cotton (Gossypium hirsutum L.) Cytoplasmic Male Sterile Line J4A.

Cytoplasmic male sterile (CMS) systems have great potential in hybrid seed production in many plants. However, the incompatibility between the cytoplasmic and nuclear genes and the availability of fewer CMS lines limit the system's application in cotton heterosis. Therefore, the present study sequenced the mitochondrial (mt) genomes of a novel cotton (Gossypium hirsutum L.) CMS line J4A and its cytoplasmic nuclear homologous maintainer line J4B to investigate the mechanism underlying CMS and improve its application. A novel CMS gene, orf116b, was identified in the CMS line. Ectopic expression of orf116b in Escherichia coli suppressed growth, while its overexpression in Arabidopsis, rice, tobacco, and cotton led to complete or partial male sterility. Further analysis of anthers revealed mt disruption in J4A with higher levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA), but lower levels of ATP and ribosomal protein (RP) than in J4B. Finally, a membrane-based yeast two-hybrid (MYTH) assay and bimolecular fluorescence complementation (BiFC) assays demonstrated that orf116b probably interacts with an anther-specific protein, LAT52. These observations collectively proved that orf116b is associated with early and stable pollen abortion in cotton, providing a foundation for further research on cotton fertility restoration and heterosis breeding.

Genome-wide identification of SAP family genes and characterization of TaSAP6-A1 to improve Cd tolerance in Triticum aestivum L.

Stress Associated Proteins (SAPs) contain A20/AN1 zinc finger domains and, have been proposed to function in various physiological processes such as cold, salinity, drought, heavy metals, damage, and flooding resistance in plants. Here, a total of 131 SAP genes were identified, including T. aestivum (60), T. urartu (10), Ae. Tauschii (16), T. dicoccoides (13), O. sativa (18), and A. thaliana (14). A phylogenetic analysis revealed that the SAPs are clustered into two subfamilies. The TaSAP genes in the collinear region comprised 34 pairs of duplicated genes formed through segmental duplication events. Overexpressing TaSAP6-A1 in wheat enhanced Cd tolerance, whereas knock-down of this gene increased Cd sensitivity. Yeast two-hybrid (Y2H) and bimolecular fluorescent complementation assays (BiFC) demonstrated interaction between TaSAP6-A1 and phenylalanine ammonia-lyase (TaPAL), the first enzyme in the phenylpropanoid pathway. This study provides a valuable reference for further investigations into the functional and molecular mechanisms of the SAP gene family.

Analysis of Rfo-Mediated Network in Regulating Fertility Restoration in Brassica oleracea.

Ogura cytoplasmic male sterility (CMS) lines play a crucial role in the utilization of heterosis. However, valuable traits, such as disease resistance genes from Ogura CMS hybrids, are challenging to incorporate for germplasm innovation, particularly in cabbage and broccoli. To date, the Rfo-mediated network regulating fertility restoration remains largely unexplored. In this study, we conducted a transcriptomic analysis of broccoli flower buds from Ogura CMS SFB45 and its Rfo-transgenic fertility restoration line, pRfo, at different stages of pollen development. Gene Ontology (GO) terms such as "pollen exine formation", "flavonoid metabolic and biosynthetic processes", and "pollen wall assembly", along with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including "flavonoid biosynthesis", "MAPK signaling pathway-plant", and "ABC transporters", were significantly enriched. We identified five differentially expressed genes (DEGs) involved in tapetum-mediated callose metabolism, thirty-four DEGs related to tapetum-mediated pollen wall formation, three DEGs regulating tapetum programmed cell death (PCD), five MPKs encoding DEGs, and twelve DEGs associated with oxidative phosphorylation. Additionally, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that RFO directly interacts with ORF138 at the protein level. These findings provide valuable insights into the fertility recovery mechanisms regulated by Rfo in broccoli and offer important clues for breeders aiming to enhance Ogura CMS hybrids in Brassica oleracea.