H2O2 negatively regulates aluminum resistance via oxidation and degradation of the transcription factor STOP1.

Aluminum (Al) stress triggers the accumulation of hydrogen peroxide (H2O2) in roots. However, whether H2O2 plays a regulatory role in aluminum resistance remains unclear. In this study, we show that H2O2 plays a crucial role in regulation of Al resistance, which is modulated by the mitochondrion-localized pentatricopeptide repeat protein REGULATION OF ALMT1 EXPRESSION 6 (RAE6). Mutation in RAE6 impairs the activity of complex I of the mitochondrial electron transport chain, resulting in the accumulation of H2O2 and increased sensitivity to Al. Our results suggest that higher H2O2 concentrations promote the oxidation of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1), an essential transcription factor that promotes Al resistance, thereby promoting its degradation by enhancing the interaction between STOP1 and the F-box protein RAE1. Conversely, decreasing H2O2 levels or blocking the oxidation of STOP1 leads to greater STOP1 stability and increased Al resistance. Moreover, we show that the thioredoxin TRX1 interacts with STOP1 to catalyze its chemical reduction. Thus, our results highlight the importance of H2O2 in Al resistance and regulation of STOP1 stability in Arabidopsis (Arabidopsis thaliana).

Rapid and Green Fabrication of Nanozyme with Geminal CuN3O Configuration for Efficient Catecholase-Mimicking.

Fabrication of nanozyme with catecholase-like catalytic activity faces the great challenge of merging outstanding activity with low cost as well as simple, rapid, and low-energy-consumed production, restricting its industrial applications. Herein, an inexpensive yet robust nanozyme (i.e., DT-Cu) via simple one-step coordination between diaminotriazole (DT) and CuSO4 within 1 h in water at room temperature is constructed. The asymmetric dicopper site with CuN3O configuration for each copper as well as Cu─O bond length of ≈1.83 Å and Cu···Cu distance of ≈3.5 Å in DT-Cu resemble those in catechol oxidase (CO), which ensure its prominent intrinsic activity, outperforming most CO-mimicking nanozymes and artificial homogeneous catalysts. The use of inexpensive DT/CuSO4 in this one-pot strategy endows DT-Cu with only ≈20% cost of natural CO per activity unit. During catalysis, O2 experienced a 4e-dominated reduction process accompanied by the formation of 1O2 and H2O2 intermediates and the product of H2O. Benefiting from the low cost as well as the distinctive structure and superior intrinsic activity, DT-Cu presents potential applications ranging from biocatalysis to analytical detection of biomolecules such as epinephrine and beyond.

PP2C.D phosphatase SAL1 positively regulates aluminum resistance via restriction of aluminum uptake in rice.

Aluminum (Al) toxicity represents a primary constraint for crop production in acidic soils. Rice (Oryza sativa) is a highly Al-resistant species; however, the molecular mechanisms underlying its high Al resistance are still not fully understood. Here, we identified SAL1 (SENSITIVE TO ALUMINUM 1), which encodes a plasma membrane (PM)-localized PP2C.D phosphatase, as a crucial regulator of Al resistance using a forward genetic screen. SAL1 was found to interact with and inhibit the activity of PM H+-ATPases, and mutation of SAL1 increased PM H+-ATPase activity and Al uptake, causing hypersensitivity to internal Al toxicity. Furthermore, knockout of NRAT1 (NRAMP ALUMINUM TRANSPORTER 1) encoding an Al uptake transporter in a sal1 background rescued the Al-sensitive phenotype of sal1, revealing that coordination of Al accumulation in the cell, wall and symplasm is critical for Al resistance in rice. By contrast, we found that mutations of PP2C.D phosphatase-encoding genes in Arabidopsis (Arabidopsis thaliana) enhanced Al resistance, which was attributed to increased malate secretion. Our results reveal the importance of PP2C.D phosphatases in Al resistance and the different strategies used by rice and Arabidopsis to defend against Al toxicity.

Development of a Risk Assessment Model for Predicting Red Blood Cell Transfusion in Neonatal Patients.

BACKGROUND: The goal was to develop a risk assessment model for predicting red blood cell (RBC) transfusion in neonatal patients to assist hospital blood supply departments in providing small portions of RBCs to those requiring RBC transfusion on time. METHODS: Clinical information was collected from 1,201 children admitted to the neonatal unit. Clinical factors associated with predicting RBC transfusion were screened, and prediction models were developed using stepwise and multifactorial logistic regression analyses, followed by the evaluation of prediction models using receiver operating characteristic curves, calibration curves, and decision curve analysis (DCA). RESULTS: Overall, 81 neonatal patients were transfused with RBCs, and the variables of gestational age at birth, age < 1 month, receipt of mechanical ventilation, and infant anemia were included in the final prediction model. The area under the curve of the prediction model was 0.936 (0.921 - 0.949), which was significantly higher than that of the individual indicators of gestational age at birth, age at admission < 1 month, receipt of mechanical ventilation, and infant anemia (p < 0.001). DCA showed a standardized net benefit for the possible risk of infant RBC transfusion at 0.1 - 1.0. CONCLUSIONS: We developed a risk assessment model to predict the risk of RBC transfusion in neonatal patients that can effectively assess the risk of RBC transfusion in children.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

Carbon Nitride-Based Heterojunction Photoelectrodes with Modulable Charge-Transfer Pathways toward Selective Biosensing.

Photoelectrochemical (PEC) sensing enables the rapid, accurate, and highly sensitive detection of biologically important chemicals. However, achieving high selectivity without external biological elements remains a challenge because the PEC reactions inherently have poor selectivity. Herein, we report a strategy to address this problem by regulating the charge-transfer pathways using polymeric carbon nitride (pCN)-based heterojunction photoelectrodes. Interestingly, because of redox reactions at different semiconductor/electrolyte interfaces with specific charge-transfer pathways, each analyte demonstrated a unique combination of photocurrent-change polarity. Based on this principle, a pCN-based PEC sensor for the highly selective sensing of ascorbic acid in serum against typical interferences, such as dopamine, glutathione, epinephrine, and citric acid was successfully developed. This study sheds light on a general PEC sensing strategy with high selectivity without biorecognition units by engineering charge-transfer pathways in heterojunctions on photoelectrodes.

Regulation of Aluminum Resistance in Arabidopsis Involves the SUMOylation of the Zinc Finger Transcription Factor STOP1.

Aluminum (Al) is a primary constraint for crop production on acid soils, which make up more than 30% of the arable land in the world. Al resistance in Arabidopsis (Arabidopsis thaliana) is achieved by malate secretion mediated by the Al-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) transporter. The C2H2-type transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) is essential and required for Al resistance, where it acts by inducing the expression of Al-resistance genes, including AtALMT1 In this study, we report that STOP1 protein function is modified by SUMOylation. The SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protease ESD4, but not other SUMO proteases, specifically interacts with and deSUMOylates STOP1. Mutation of ESD4 increases the level of STOP1 SUMOylation and the expression of the STOP1-regulated gene AtALMT1, which contributes to the increased Al resistance in esd4 The esd4 mutation does not influence STOP1 protein abundance but increases the association of STOP1 with the AtALMT1 promoter, which might explain the elevated expression of AtALMT1 in esd4 We demonstrate that STOP1 is mono-SUMOylated at K40, K212, or K395 sites, and blocking STOP1 SUMOylation reduces STOP1 stability and the expression of STOP1-regulated genes, leading to the reduced Al resistance. Our results thus reveal the involvement of SUMOylation in the regulation of STOP1 and Al resistance in Arabidopsis.

Degradation of STOP1 mediated by the F-box proteins RAH1 and RAE1 balances aluminum resistance and plant growth in Arabidopsis thaliana.

The C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) is crucial for aluminum (Al) resistance in Arabidopsis. The F-box protein Regulation of AtALMT1 Expression 1 (RAE1) was recently reported to regulate the stability of STOP1. There is a unique homolog of RAE1, RAH1 (RAE1 homolog 1), in Arabidopsis, but the biological function of RAH1 is still not known. In this study, we characterize the role of RAH1 and/or RAE1 in the regulation of Al resistance and plant growth. We demonstrate that RAH1 can directly interact with STOP1 and promote its ubiquitination and degradation. RAH1 is preferentially expressed in root caps and various vascular tissues, and its expression is induced by Al and controlled by STOP1. Mutation of RAH1 in rae1 but not the wild-type (WT) background increases the level of STOP1 protein, leading to increased expression of STOP1-regulated genes and enhanced Al resistance. Interestingly, the rah1rae1 double mutant shows reduced plant growth compared with the WT and single mutants under normal conditions, and introduction of stop1 mutation into the double mutant background can rescue its reduced plant growth phenotype. Our results thus reveal that RAH1 plays an unequally redundant role with RAE1 in the modulation of STOP1 stability and plant growth, and dynamic regulation of the STOP1 level is critical for the balance of Al resistance and normal plant growth.

Calmodulin-like protein CML24 interacts with CAMTA2 and WRKY46 to regulate ALMT1-dependent Al resistance in Arabidopsis thaliana.

ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1)-mediated malate exudation from roots is critical for aluminium (Al) resistance in Arabidopsis. Its upstream molecular signalling regulation is not yet well understood. The role of CALMODULIN-LIKE24 (CML24) in Al-inhibited root growth and downstream molecular regulation of ALMT1-meditaed Al resistance was investigated. CML24 confers Al resistance demonstrated by an increased root-growth inhibition of the cml24 loss-of-function mutant under Al stress. This occurs mainly through the regulation of the ALMT1-mediated malate exudation from roots. The mutation and overexpression of CML24 leads to an elevated and reduced Al accumulation in the cell wall of roots, respectively. Al stress induced both transcript and protein abundance of CML24 in root tips, especially in the transition zone. CML24 interacts with CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2 (CAMTA2) and promotes its transcriptional activity in the regulation of ALMT1 expression. This results in an enhanced malate exudation from roots and less root-growth inhibition under Al stress. Both CML24 and CAMTA2 interacted with WRKY46 suppressing the transcriptional repression of ALMT1 by WRKY46. The study provides novel insights into understanding of the upstream molecular signalling of the ALMT1-depdendent Al resistance.

F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis.

Aluminum (Al) toxicity is a major factor limiting crop production on acid soils, which represent over 30% of the world's arable land. Some plants have evolved mechanisms to detoxify Al. Arabidopsis, for example, secretes malate via the AtALMT1 transporter to chelate and detoxify Al. The C2H2-type transcription factor STOP1 plays a crucial role in Al resistance by inducing the expression of a set of genes, including AtALMT1 Here, we identify and characterize an F-box protein-encoding gene regulation of Atalmt1 expression 1 (RAE1) that regulates the level of STOP1. Mutation and overexpression of RAE1 increases or decreases the expression of AtALMT1 and other STOP1-regulated genes, respectively. RAE1 interacts with and promotes the degradation of STOP1 via the ubiquitin-26S proteasome pathway, while Al stress promotes the accumulation of STOP1. We find that STOP1 up-regulates RAE1 expression by directly binding to the RAE1 promoter, thus forming a negative feedback loop between STOP1 and RAE1. Our results demonstrate that RAE1 influences Al resistance through the ubiquitination and degradation of STOP1.

Dysfunction of the 4-coumarate:coenzyme A ligase 4CL4 impacts aluminum resistance and lignin accumulation in rice.

The root cell wall is the first and primary target of aluminum (Al) toxicity. Monocots such as rice (Oryza sativa) can accumulate appreciable levels of hydroxycinnamic acids (HCAs) to modify and cross-link hemicellulose and/or lignin of the cell wall. Nevertheless, it is unclear whether this HCA-mediated modification of the cell wall is important for Al accumulation and resistance. We previously isolated and characterized a rice ral1 (resistance to aluminum 1) mutant that shows enhanced Al resistance. In this study, we cloned RAL1 and found that it encodes the 4-coumarate:coenzyme A ligase 4CL4, an enzyme putatively involved in lignin biosynthesis. Mutation of RAL1/4CL4 reduces lignin content and increases the accumulation of its substrates 4-coumaric acid (PA) and ferulic acid (FA). We demonstrate that altered lignin accumulation is not required for the enhanced Al resistance in ral1/4cl4 mutants. We found that the increased accumulation of PA and FA can reduce Al binding to hemicellulose and consequently enhance Al resistance in ral1/4cl4 mutants. Al stress is able to trigger PA and FA accumulation, which is likely caused by the repression of the expression of RAL1/4CL4 and its homologous genes. Our results thus reveal that Al-induced PA and FA accumulation is actively and positively involved in Al resistance in rice through the modification of the cell wall and thereby the reduced Al binding to the cell wall.

Golgi-localised manganese transporter PML3 regulates Arabidopsis growth through modulating Golgi glycosylation and cell wall biosynthesis.

Golgi is a critical compartment for both the reutilisation of the essential micronutrient manganese (Mn) and its detoxification. However, whether Mn plays a role in the Golgi remains to be demonstrated in plants. We characterised the function of PML3, a member of the Unknown Protein Family UPF0016, in Mn transport and the regulation of plant growth, Golgi glycosylation and cell wall biosynthesis in Arabidopsis. We also investigated the relationship of PML3 with NRAMP2, a trans-Golgi network localised Mn transporter. PML3-GFP is preferentially localised in the cis-Golgi. PML3 can transport Mn to rescue the hypersensitivity of yeast mutant Δpmr1 to excess Mn. Two mutant alleles of PML3 displayed reduced plant growth and impaired seed development under Mn-deficient conditions. The pml3 mutants also showed impaired Golgi glycosylation and cell wall biosynthesis under Mn deficiency. Double mutations of PML3 and NRAMP2 showed improved plant growth compared with that of single mutants under Mn deficiency, implying that PML3 and NRAMP2 play opposite roles in the regulation of Golgi Mn levels. Our results suggest that PML3 mediates Mn uptake into the Golgi compartments, which is required for proper protein glycosylation and cell wall biosynthesis under Mn-deficient conditions.

DNA demethylase ROS1 negatively regulates the imprinting of DOGL4 and seed dormancy in Arabidopsis thaliana.

Genomic imprinting is a form of epigenetic regulation resulting in differential gene expression that reflects the parent of origin. In plants, imprinted gene expression predominantly occurs in the seed endosperm. Maternal-specific DNA demethylation by the DNA demethylase DME frequently underlies genomic imprinting in endosperm. Whether other more ubiquitously expressed DNA demethylases regulate imprinting is unknown. Here, we found that the DNA demethylase ROS1 regulates the imprinting of DOGL4DOGL4 is expressed from the maternal allele in endosperm and displays preferential methylation and suppression of the paternal allele. We found that ROS1 negatively regulates imprinting by demethylating the paternal allele, preventing its hypermethylation and complete silencing. Furthermore, we found that DOGL4 negatively affects seed dormancy and response to the phytohormone abscisic acid and that ROS1 controls these processes by regulating DOGL4 Our results reveal roles for ROS1 in mitigating imprinted gene expression and regulating seed dormancy.

Roles of DEMETER in regulating DNA methylation in vegetative tissues and pathogen resistance.

DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5-methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss-of-function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell-specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

Mutation of HPR1 encoding a component of the THO/TREX complex reduces STOP1 accumulation and aluminium resistance in Arabidopsis thaliana.

C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) plays an essential role in aluminium (Al) resistance in Arabidopsis thaliana by controlling the expression of a set of Al-resistance genes, including the malate transporter-encoding gene A. thaliana aluminium activated malate transporter 1 (AtALMT1) that is critically required for Al resistance. STOP1 is suggested to be modulated by Al at post-transcriptional and/or post-translational levels. However, the underlying molecular mechanisms remain to be demonstrated. We carried out a forward genetic screen on an ethyl methanesulphonate mutagenized population, which contains the AtALMT1 promoter-driven luciferase reporter gene (pAtALMT1:LUC), and identified hyperrecombination protein 1 (HPR1), which encodes a subunit of the THO/TREX complex. We investigate the effect of hpr1 mutations on the expression of Al-resistance genes and Al resistance, and we also examined the regulatory role of HPR1 in nuclear messenger RNA (mRNA) and protein accumulation of STOP1 gene. Mutation of HPR1 reduces the expression of STOP1-regulated genes and the associated Al resistance. The hpr1 mutations increase STOP1 mRNA retention in the nucleus and consequently decrease STOP1 protein abundance. Mutation of regulation of AtALMT1 expression 1 (RAE1) that mediates STOP1 degradation in the hpr1 mutant background can partially rescue the deficient phenotypes of hpr1 mutants. Our results demonstrate that HPR1 modulates Al resistance partly through the regulation of nucleocytoplasmic STOP1 mRNA export.

Natural variation in the promoter of OsHMA3 contributes to differential grain cadmium accumulation between Indica and Japonica rice.

Rice is a major source of cadmium (Cd) intake for Asian people. Indica rice usually accumulates more Cd in shoots and grains than Japonica rice. However, underlying genetic bases for differential Cd accumulation between Indica and Japonica rice are still unknown. In this study, we cloned a quantitative trait locus (QTL) grain Cd concentration on chromosome 7 (GCC7) responsible for differential grain Cd accumulation between two rice varieties by performing QTL analysis and map-based cloning. We found that the two GCC7 alleles, GCC7PA64s and GCC793-11 , had different promoter activity of OsHMA3, leading to different OsHMA3 expression and different shoot and grain Cd concentrations. By analyzing the distribution of different haplotypes of GCC7 among diverse rice accessions, we discovered that the high and low Cd accumulation alleles, namely GCC793-11 and GCC7PA64s , were preferentially distributed in Indica and Japonica rice, respectively. We further showed that the GCC7PA64s allele can be used to replace the GCC793-11 allele in the super cultivar 93-11 to reduce grain Cd concentration without adverse effect on agronomic traits. Our results thus reveal that the QTL GCC7 with sequence variation in the OsHMA3 promoter is an important determinant controlling differential grain Cd accumulation between Indica and Japonica rice.

NRAMP2, a trans-Golgi network-localized manganese transporter, is required for Arabidopsis root growth under manganese deficiency.

To cope with manganese (Mn) deficiency, plants have evolved an efficient transport system to uptake and redistribute Mn. However, the underlying molecular mechanisms remain to be demonstrated. We carried out a forward genetic screen in a root high-affinity Mn transporter nramp1 mutant background in Arabidopsis thaliana and identified an uncharacterized Mn transport NRAMP2. We investigated the effect of nramp2 mutation on root growth and reactive oxygen species (ROS) accumulation and we also examined the NRAMP2 expression pattern, and the subcellular localization and transport activity of NRAMP2. Mutation of NRAMP2 impaired plant growth, while overexpression of NRAMP2 improved plant growth under low Mn conditions. In the nramp2-1nramp1 double mutant, Mn deficiency inhibited root cell elongation and root hair development, which was associated with increased hydrogen peroxide (H2 O2 ) accumulation. NRAMP2 is preferentially localized to the trans-Golgi network. NRAMP2 has Mn influx transport activity in yeast, and mutation of NRAMP2 led to greater Mn retention in roots. Our results suggest that under Mn-deficient conditions, increased accumulation of H2 O2 is partially responsible for the root growth inhibition and NRAMP2 is involved in remobilization of Mn in Golgi for root growth.

A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars.

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non-functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380(th) position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F1 test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root-to-shoot Cd translocation and a shift in root Cd speciation from Cd-S to Cd-O bonding determined by synchrotron X-ray absorption spectroscopy. Our study has identified a new loss-of-function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.