Background Ischemia–reperfusion–induced acute kidney injury (I/R-AKI) causes profound bioenergetic collapse in renal proximal tubular epithelial cells, triggering a sustained shift from mitochondrial oxidative metabolism to glycolysis. Although lactate accumulation is a hallmark of this metabolic state, whether lactate actively drives mitochondrial dysfunction and enforces persistent metabolic reprogramming remains unclear. Methods Human renal biopsy specimens, murine I/R-AKI, and hypoxia/reoxygenation–challenged proximal tubular epithelial cells were used to investigate the lactate–MRS2 axis in I/R-AKI. Lactate signaling was inhibited by sodium oxamate, while MRS2 was suppressed using CPACC or siRNA-based approaches, including lipid nanoparticle–mediated siMRS2 delivery. Mitochondrial function and oxidative metabolism were assessed by oxygen consumption rate, ATP production, mitochondrial membrane potential, and tricarboxylic acid cycle (TCA) gene expression. Results I/R-AKI induced a pronounced bioenergetic deficit in proximal tubules, marked by disrupted mitochondrial homeostasis, suppressed TCA cycle activity and enhanced aerobic glycolysis. Glycolysis-derived lactate accumulated during reperfusion, disrupting mitochondrial oxidative metabolism, whereas inhibition of lactate production with sodium oxamate attenuated tubular injury and restored mitochondrial metabolic function. Mechanistically, lactate activated the mitochondrial Mg²⁺ channel MRS2, causing mitochondrial Mg²⁺ overload and sustained reliance on inefficient glycolysis. Targeting MRS2, either pharmacologically or via lipid nanoparticle–mediated siRNA delivery, normalized mitochondrial Mg²⁺ homeostasis, improved mitochondrial function and reinstated oxidative metabolism following I/R-AKI. Conclusion This study identifies a previously unrecognized lactate–MRS2 signaling axis that drives maladaptive metabolic reprogramming in ischemic AKI. Targeting MRS2 to restore mitochondrial Mg²⁺ homeostasis reinstates oxidative metabolism, breaks maladaptive metabolic reprogramming, and promotes renal recovery.

Podocyte injury associated with albuminuria and diabetic nephropathy (DN) progression. N6-methyladenosine (m6A) is a common form of epigenetic modification in eukaryotic cells and is known to be associated with a variety of disease processes. Its role in podocyte injury of DN remains poorly studied. We observed a higher expression of fat mass and obesity-associated protein (FTO) both in diabetic mice and human kidneys and DN podocytes in vitro, and the level of FTO was correlated with lipid accumulation. Furthermore, we confirmed that two selective FTO demethylation inhibitors meclofenamic acid (MA) and diacerein (DIA) administration effectively ameliorated lipotoxicity-induced podocyte injury, evidenced by restored autophagy, inhibition of apoptosis and inflammation, as well as mitigating endoplasmic reticulum stress (ERS) and mitochondrial damage both in vitro and vivo model of DN. Mechanistically, FTO demethylation inhibitors downregulated Acetyl-CoAcarboxylase 1 (ACC1) levels in db/db mice and advanced glycation end product (AGE)-treated podocytes, subsequently decreased podocyte fatty acid accumulation. ACC1 was identified as a direct FTO target in which FTO stabilizes ACC1 mRNA with the mediation of YTH domain-containing family protein 2 (YTHDF2) in an m6Adependent manner using m6A RNA immunoprecipitation-quantitative real-time PCR (MeRIP-qPCR) and dualluciferase reporter gene assays. Collectively, our findings demonstrate an important role of FTO mediatedm6A modification of ACC1 contributed to s lipotoxicity-mediated injury of DN podocytes, which provide fresh insights into the therapeutic strategies for DN.

Chaperone-mediated autophagy (CMA) is a specific form of autophagy that selectively targets proteins containing a KFERQ-like motif and relies on the chaperone protein HSPA8/HSC70 for substrate recognition. In SERPINA1/a1-antitrypsin deficiency (AATD), a disease characterized by the hepatic buildup of the SERPINA1E342K/ATZ, CMA's role had been unclear. This work demonstrates the critical role that CMA plays in preventing SERPINA1E342K/ATZ accumulation; suppressing CMA worsens SERPINA1E342K/ATZ accumulation while activating it through chemical stimulation or LAMP2A overexpression promotes SERPINA1E342K/ATZ breakdown. Specifically, SERPINA1E342K/ATZ's 121QELLR125 motif is critical for HSPA8/HSC70 recognition and LAMP2A's charged C-terminal cytoplasmic tail is vital for substrate binding, facilitating CMA-mediated degradation of SERPINA1E342K/ATZ. This selective activation of CMA operates independently of other autophagy pathways and alleviates SERPINA1E342K/ATZ aggregate-induced cellular stress. In vivo administration of AR7 promotes hepatic SERPINA1E342K/ATZ elimination and mitigates hepatic SERPINA1E342K/ATZ aggregation pathology. These findings highlight CMA's critical function in cellular protein quality control of SERPINA1E342K/ATZ and place it as a novel target for AATD treatment.Abbreviation: AR7: atypical retinoid 7; ATG16L1: autophagy related 16 like 1; AATD: SERPINA1/alpha-1 antitrypsin deficiency; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; LAMP2B: lysosomal associated membrane protein 2B; LAMP2C: lysosomal associated membrane protein 2C; MG132: carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; PAS-D: periodic acid-Schiff plus diastase; SERPINA1/A1AT: serpin family A member 1; SERPINA1E342K/ATZ: Z variant of SERPINA1; TMRE: tetramethyl rhodamine ethyl ester perchlorate.

Pecan [Carya illinoinensis (Wangenh.) Koch] is a widely cultivated dried fruit and woody oil tree with high economic value. Continuous sole planting of pecan caused some land to lie idle and deterioration of soil conditions. Tree and medicinal crops intercropping represents an environmental-friendly and economically feasible solution to these issues. Thus, we aimed to explore the underlying mechanism by which intercropping improved soil condition by regulating the interactions of the soil microbiome and metabolome. In this study, pecans were intercropped with medicinal crops caper spurge and honeysuckle on a tree farm in China. A combined analysis of soil microbiomes and metabolomes was performed to discover the effects of intercropping on bulk and rhizosphere soils. The results showed that intercropping improved the edaphic properties of bulk soil and promoted the growth of pecan and caper spurge. Intercropping also significantly altered the structures of both bacterial and fungal communities in bulk soil, stabilised the enrichment of nitrogen-cyclic bacteria, for instance, Bacillus, and decreased the relative abundances of plant–pathogenic fungi, for instance, Fusarium. In addition, the result of metabolomic analysis showed that intercropping promoted the synthesis of functional compounds, such as trehalose and ethanolamine, which enhanced plant disease resistance in bulk soils. Moreover, the co-occurrence networks of microbiomes and metabolomes of bulk soils revealed that Bacillus was significantly correlated with Fusarium, Alternaria, and trehalose under intercropping patterns. Furthermore, analysis of microbiomes and metabolomes in the rhizosphere soil of caper spurge and honeysuckle revealed that Penicillium and Rhizomicrobium were significantly increased by intercropping and showed more dynamic connections with other genera and metabolites compared with single planting. Overall, intercropping pecans with caper spurge and honeysuckle can improve soil conditions and promote plant growth through microbiological and metabolomics integrated analyses. This study provides valuable information and theoretical basis for optimizing land resource utilisation and improving soil conditions in tree fields like pecan fields via intercropping, thereby reducing production losses and ensuring economic benefits.

miRNAs function as negative regulators that significantly influence plant growth and stress responses. Within rice and other monocotyledonous plants, miR1432 plays a conserved role in seed development and disease resistance. However, its involvement in the response to abiotic stresses remains unclear. Our study aimed to elucidate this mechanism by predicting the targeting of the rice P-type IIB Ca2+ ATPase gene OsACAs by miR1432 and identifying its cleavage sites via 5 ' RACE. We observed induced expression of miR1432 and its target gene, OsACA6, under abiotic stresses. Overexpression (OX) of miR1432 and suppression of OsACA6 resulted in reduced cold, salt, and drought tolerance, while OsACA6 suppression/knockout and OX had opposite effects on cold tolerance. Additionally, miR1432 may target other OsACA6 homologs. RNA-sequencing data highlighted the differential expression of stress-related genes in miR1432-overexpressing rice. Furthermore, miR1432-overexpressing rice exhibited weakened vigor, dwarfism, yellowing leaves and reduced fertility. Collectively, our results strongly suggest that miR1432 not only negatively modulates abiotic stress tolerance by suppressing Ca2+ ATPase gene(s) but also influences plant growth and development.

Histone citrullination, an important post-translational modification mediated by peptidyl arginine deiminases, is essential for many physiological processes and epigenetic regulation. However, the causal relationship between histone citrullination and specific gene regulation remains unresolved. In this study, we develop a programmable epigenetic editor by fusing the peptidyl arginine deiminase PPAD from Porphyromonas gingivalis with dCas9. With the assistance of gRNA, PPAD-dCas9 can recruit peptidyl arginine deiminases to specific genomic loci, enabling direct manipulation of the epigenetic landscape and regulation of gene expression. Our citrullination editor allows for site-specific manipulation of histone H3R2,8,17 and 26 at target human gene loci, resulting in the activation or suppression of different genes in a locus-specific manner. Moreover, the epigenetic effects of the citrullination editor are specific and sustained. This epigenetic editor offers an accurate and efficient tool for exploring gene regulation of histone citrullination.

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.

The spike (S) protein plays a crucial role in the entry of SARS-CoV-2 into host cells. The S protein contains two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit binds to the receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells. Therefore, degrading S1 is one of the feasible strategies to inhibit SARS-CoV-2 infection. The purpose of this study is to develop a degradation tool targeting S1. First, we constructed a HEK 293 cell line stably expressing S1 by using a three-plasmid lentivirus system. The overexpression of the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) in this cell line promoted the ubiquitination of S1 and accelerated its proteasomal degradation. Further research showed the polyubiquitination of S1 catalyzed by MUL1 mainly occurred via the addition of K48-linked chains. Moreover, the specific peptide LCB1, which targets and recognizes S1, was combined with MUL1 to create the chimeric E3 ubiquitin ligase LCB1-MUL1. In comparison to MUL1, this chimeric enzyme demonstrated improved catalytic efficiency, resulting in a reduction of S1's half-life from 12 h to 9 h. In summary, this study elucidated the mechanism by which MUL1 promotes the ubiquitination modification of S1 and facilitates its degradation through the proteasome, and preliminarily validated the effectiveness of targeted degradation of S1 by chimeric enzyme LCB1-MUL1.

Chaperone-mediated autophagy (CMA) is a specific form of autophagy that selectively targets proteins containing a KFERQ-like motif and relies on the chaperone protein HSC70 for substrate recognition. In α1-antitrypsin deficiency (AATD), a disease characterized by the hepatic build-up of α1-Antitrypsin Z mutant (ATZ), CMA’s role had been unclear. This work demonstrates the critical role that CMA plays in preventing ATZ accumulation; suppressing CMA worsens ATZ accumulation, whilst activating it through chemical stimulation or LAMP2A overexpression promotes ATZ breakdown. Specifically, ATZ’s 121QELLR125 motif is critical for HSC70 recognition and LAMP2A’s charged C-terminal cytoplasmic tail is vital for substrate binding, facilitating CMA-mediated degradation of ATZ. This selective activation of CMA operates independently from other autophagy pathways and alleviate ATZ aggregates caused cellular stress. These findings highlight CMA’s critical function in cellular protein quality control of ATZ and place it as a novel target for AATD treatment approaches.

Diabetic nephropathy (DN) contributes to increased morbidity and mortality among patients with diabetes and presents a considerable global health challenge. However, reliable biomarkers of DN have not yet been established. Phosphorylated proteins are crucial for disease progression. However, their diagnostic potential remains unexplored. In this study, we used ultra-high-sensitivity quantitative phosphoproteomics to identify phosphoproteins in urinary extracellular vesicles (uEVs) as potential biomarkers of DN. We detected 233 phosphopeptides within the uEVs, with 47 phosphoproteins exhibiting significant alterations in patients with DN compared to those in patients with diabetes. From these phosphoproteins, we selected phosphorylated aquaporin-2 (p-AQP2[S256]) and phosphorylated glycogen synthase kinase-3β (p-GSK3β[Y216]) for validation, as they were significantly overrepresented in pathway analyses and previously implicated in DN pathogenesis. Both phosphoproteins were successfully confirmed through Phos-tag western blotting in uEVs and immunohistochemistry staining in kidney sections, suggesting that phosphoprotein alterations in uEVs reflect corresponding changes within the kidney and their potential as candidate biomarkers for DN. Our research proposes the utilization of phosphoproteins in uEVs as a liquid biopsy, presenting a highly feasible diagnostic tool for kidney disease.

TWIK-related acid-sensitive K+ channel-2 (TASK-2, encoded by Kcnk5) is essential in cell biological processes, by regulating transmembrane K+ balance. In the present study, we aimed to clarify the role of TASK-2 in renal fibrosis and explore the underlying mechanism. We found that TASK-2 level was elevated in the renal tubular UUO- and UIR-induced renal fibrosis as well as in patients with renal tubulointerstitial fibrosis. Knockout of Kcnk5 or inhibition of TASK-2 in renal tubules attenuated G2/M cell-cycle arrest and alleviated renal fibrosis. Mechanistically, demethylase fat mass and obesity-associated protein (FTO) reduced N6-adenosine methylation (m6A) of Kcnk5 mRNA following renal fibrosis. FTO deficiency attenuated the upregulation of TASK-2 and renal fibrosis. The results demonstrated the crucial role of TASK-2 in renal fibrosis, which is conducive to a better understanding of the pathogenesis of renal fibrosis. TASK-2 may be a potential treatment strategy to alleviate the development of renal fibrosis.

Heterosis, an important biological phenomenon wherein F1 hybrids exhibit better performance than any of their parents, has been widely applied; however, its underlying mechanism remains largely unknown. Here, we studied and compared the dynamic transcriptional profiles of super-hybrid rice LY2186 and its parents at 17 time points during 2 day/night cycles and identified 1552 rhythmic differentially expressed genes (RDGs). Cluster and functional enrichment analyses revealed that the day- and night-phased RDGs were mainly enriched in the photosynthesis and stress response categories, respectively. Regulatory network analysis indicated that circadian-related RDGs are core components in both the day and night phases and extensively regulate downstream genes involved in photosynthesis, starch synthesis, plant hormone signal transduction, and other pathways. Furthermore, among the 282 RDGs mapped onto the quantitative tract loci of small intervals (≤100 genes), 72.3% were significantly enriched in the yield, vigor, and anatomy categories. These findings provide valuable information for exploring heterosis mechanisms further and guiding breeding practices.

Kidney fibrosis is a common process that leads to the progression of various types of kidney disease. We used an integrated computational and experimental systems biology approach to identify protein kinases that regulate gene expression changes in the kidneys of human immunodeficiency virus (HIV) transgenic mice (Tg26 mice), which have both tubulointerstitial fibrosis and glomerulosclerosis. We identified homeo-domain interacting protein kinase 2 (HIPK2) as a key regulator of kidney fibrosis. HIPK2 was upregulated in the kidneys of Tg26 mice and in those of patients with various kidney diseases. HIV infection increased the protein concentrations of HIPK2 by promoting oxidative stress, which inhibited the seven in absentia homolog 1 (SIAH1)-mediated proteasomal degradation of HIPK2. HIPK2 induced apoptosis and the expression of epithelial-to-mesenchymal transition markers in kidney epithelial cells by activating the p53, transforming growth factor β (TGF-β)-SMAD family member 3 (Smad3) and Wnt-Notch pathways. Knockout of HIPK2 improved renal function and attenuated proteinuria and kidney fibrosis in Tg26 mice, as well as in other murine models of kidney fibrosis. We therefore conclude that HIPK2 is a potential target for anti-fibrosis therapy.

Agrobacterium-mediated plant transformation has been widely used for introducing transgene(s) into a plant genome and plant breeding. However, our understanding of T-DNA integration into rice genome remains limited relative to that in the model dicot Arabidopsis. To better elucidate the T-DNA integration into the rice genome, we investigated extensively the T-DNA ends and their flanking rice genomic sequences from two transgenic rice plants carrying Cowpea Trypsin Inhibitor (CpTI)-derived gene Signal-CpTI-KDEL (SCK) and Bacillus thuringiensis (BT) gene, respectively, by TAIL-PCR method. Analysis of the junction sequences between the T-DNA ends and rice genome DNA indicated that there were three joining patterns of microhomology, filler DNA sequences, and exact joining, and both the T-DNA ends tend to adopt identical manner to join the rice genome. After T-DNA integration, there were several variations of rice genomic sequences, including small deletions at the integration sites, superfluous DNA inserted between T-DNA and genome, and translocation of genomic DNA in the flanking regions. The translocation block could be from a noncontiguous region in the same chromosome or different chromosomes at the integration sites, and the originating position of the translocated block resulted in comparable deletion based on a cut/paste mechanism rather than a replication mechanism. Our study may lead to a better understand of T-DNA integration mechanism and facilitate functional genomic studies and further crop improvement.

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). MiR-34a, a p53-regulated miRNA, directly targets SIRT1 and contributed to DN progression. MiR-34a represses SIRT1 to activate p53 and establish a positive feedback loop. However, whether p53/miR-34a/SIRT1 signaling is activated in podocytes and contributes to DN pathogenesis remains elusive. In this study, we observed that serum miR-34a level was positively correlated with podocyte injury in DN patients. The expression of acetylated p53 and miR-34a was upregulated, SIRT1was downregulated in glomeruli from patients with DN and STZ induced diabetic mice, as well as in human podocytes treated with advanced glycation end (AGE). MiR-34a antagonism in vitro and vivo in STZ induced diabetic mice developed alleviated glomerulus injury as reflected by attenuated albuminuria, reduced podocyte loss and restored autophagic flux. In human podocyte, inhibition of AGE formation by pyridoxamine prevented miR-34a dependent repression of SIRT1, p53 acetylation and activate podocyte autophagy in a dose-dependent manner. MiR-34a overexpression increases acetylation of p53 by translational repression of SIRT1. SIRT1 overexpression also impacts AGE induced apoptosis through deacetylating p53, whereas silencing of SIRT1 by EX527 attenuated the cytoprotective functions of miR-34a knockdown. Moreover, blockade of p53 acetylation significantly rescued miR-34a-induced apoptosis through SIRT1 restoration. Collectively, we demonstrate that by activation of p53, AGE induced the transcription of miR-34a, miR-34a in turn repressed SIRT1 to activate p53, resulting in a positive-feedback loop and contributing to podocyte injury. Targeting modulation of p53/miR-34a/SIRT1 feedback by miR-34a knockdown or overexpression of SIRT1 could rescue podocyte injury during DN.

Podocytes are known to play a determining role in the progression of proteinuric kidney disease. N 6 -methyladenosine (m6A), as the most abundant chemical modification in eukaryotic mRNA, has been reported to participate in various pathological processes. However, its role in podocyte injury remains unclear. In this study, we observed the elevated m6A RNA levels and the most upregulated METTL14 expression in kidneys of mice with adriamycin (ADR) and diabetic nephropathy. METTL14 was also evidently increased in renal biopsy samples from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy and in cultured human podocytes with ADR or advanced glycation end product (AGE) treatment in vitro. Functionally, we generated mice with podocyte-specific METTL14 deletion, and identified METTL14 knockout in podocytes improved glomerular function and alleviated podocyte injury, characterized by activation of autophagy and inhibition of apoptosis and inflammation, in mice with ADR nephropathy. Similar to the results in vivo, knockdown of METTL14 facilitated autophagy and alleviated apoptosis and inflammation in podocytes under ADR or AGE condition in vitro. Mechanically, we identified METTL14 knockdown upregulated the level of Sirt1, a well-known protective deacetylase in proteinuric kidney diseases, in podocytes with ADR or AGE treatment. The results of MeRIP-qPCR and dual-luciferase reporter assay indicated METTL14 promoted Sirt1 mRNA m6A modification and degradation in injured podocytes. Our findings suggest METTL14-dependent RNA m6A modification contributes to podocyte injury through posttranscriptional regulation of Sirt1 mRNA, which provide a potential approach for the diagnosis and treatment of podocytopathies.

It has been reported that Cavin1 deficiency causes lipodystrophy in both humans and mice by affecting lipid metabolism. The ablation of Cavin1 in rodents also causes a significant deviation from Mendelian ratio at weaning in a background-dependent manner, suggesting the presence of undiscovered functions of Cavin1. In the current study, the results show that Cavin1 deficiency causes neonatal death in C57BL/6J mice by dampening the storage and mobilization of glycogen in the liver, which leads to lethal neonatal hypoglycemia. Further investigation by electron microscopy reveals that Cavin1 deficiency impairs the fenestration in liver sinusoidal endothelial cells (LSECs) and impacts the permeability of endothelial barrier in the liver. Mechanistically, Cavin1 deficiency inhibits the RhoA-Rho-associated protein kinase 2-LIM domain kinase-Cofilin signaling pathway and suppresses the dynamics of the cytoskeleton, and eventually causes the reduction of fenestrae in LSECs. In addition, the defect of fenestration in LSECs caused by Cavin1 deficiency can be rescued by treatment with the F-actin depolymerization reagent latrunculin A. In summary, the current study reveals a novel function of Cavin1 on fenestrae formation in LSECs and liver glycogen metabolism, which provide an explanation for the neonatal death of Cavin1 null mice and a potential mechanism for metabolic disorders in patients with Cavin1 mutation.

Jasmonate ZIM-domain (JAZ) proteins belong to the subgroup of TIFY family and act as key regulators of jasmonate (JA) responses in plants. To date, only a few JAZ proteins have been characterized in rice. Here, we report the identification and function of rice OsJAZ13 gene. The gene encodes three different splice variants: OsJAZ13a, OsJAZ13b, and OsJAZ13c. The expression of OsJAZ13 was mainly activated in vegetative tissues and transiently responded to JA and ethylene. Subcellular localization analysis indicated OsJAZ13a is a nuclear protein. Yeast two-hybrid assays revealed OsJAZ13a directly interacts with OsMYC2, and also with OsCOI1, in a COR-dependent manner. Furthermore, OsJAZ13a recruited a general co-repressor OsTPL via an adaptor protein OsNINJA. Remarkably, overexpression of OsJAZ13a resulted in the attenuation of root by methyl JA. Furthermore, OsJAZ13a-overexpressing plants developed lesion mimics in the sheath after approximately 30–45 days of growth. Tillers with necrosis died a few days later. Gene-expression analysis suggested the role of OsJAZ13 in modulating the expression of JA/ethylene response-related genes to regulate growth and activate hypersensitive cell death. Taken together, these observations describe a novel regulatory mechanism in rice and provide the basis for elucidating the function of OsJAZ13 in signal transduction and cell death in plants.

Objectives In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. Results We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T-0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T-1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). Conclusion Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.