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).

Molecular and phylogenetic evidence of parallel expansion of anion channels in plants.

Aluminum-activated malate transporters (ALMTs) and slow anion channels (SLACs) are important in various physiological processes in plants, including stomatal regulation, nutrient uptake, and in response to abiotic stress such as aluminum toxicity. To understand their evolutionary history and functional divergence, we conducted phylogenetic and expression analyses of ALMTs and SLACs in green plants. Our findings from phylogenetic studies indicate that ALMTs and SLACs may have originated from green algae and red algae, respectively. The ALMTs of early land plants and charophytes formed a monophyletic clade consisting of three subgroups. A single duplication event of ALMTs was identified in vascular plants and subsequent duplications into six clades occurred in angiosperms, including an identified clade, 1-1. The ALMTs experienced gene number losses in clades 1-1 and 2-1 and expansions in clades 1-2 and 2-2b. Interestingly, the expansion of clade 1-2 was also associated with higher expression levels compared to genes in clades that experienced apparent loss. SLACs first diversified in bryophytes, followed by duplication in vascular plants, giving rise to three distinct clades (I, II, and III), and clade II potentially associated with stomatal control in seed plants. SLACs show losses in clades II and III without substantial expansion in clade I. Additionally, ALMT clade 2-2 and SLAC clade III contain genes specifically expressed in reproductive organs and roots in angiosperms, lycophytes, and mosses, indicating neofunctionalization. In summary, our study demonstrates the evolutionary complexity of ALMTs and SLACs, highlighting their crucial role in the adaptation and diversification of vascular plants.

A fluorescence turn "on-off" imaging probe for sequential detection of Al3+ and L-Cysteine in HeLa cells.

At this "Aluminum Age", exposure to aluminum (metallic or ionic form) is inevitable and inestimable. The presence of aluminum in biological systems is evident but more often aluminum toxicity is less understood. Therefore, the presence of biologically reactive aluminum needs to be identified and quantified. Alongside metals, L-cysteine, an essential amino acid, plays a pivotal role in the homeostasis of cellular oxidative and reductive stress. However, excess (<7g) could be lethal and can lead to death. Thus, in-situ selective detection of aluminum and L-cysteine is of larger interest. Here we report a fluorogenic probe (R) for the sequential selective detection and quantification of Al3+ and L-cysteine in a semi-aqueous medium (3:7; water: DMSO). The probe (R) was synthesized by a one-step acid-mediated condensation reaction between pyridine-3,4-diamine and 2-hydroxy-1-napthaldehyde. The synthesized probe was characterized using 1H and 13C NMR, and HR-Mass spectroscopic techniques. The probe (R) is non-emissive in nature, but on recognition of Al3+, the probe R showed "turn-on" emission (bright yellow colour) showing two emission maxima (522 nm and 547 nm), and no naked eye observable color change. Other competing cations do not show any noticeable fluorescence outcome. The R + Al3+ ensemble can specifically detect L-cysteine among all the essential amino acids by showing a fluorescence "turn-off" response. The sensing mechanism of Al3+ is obeying the chelation-enhanced fluorescence (CHEF) effect. The binding constant of R + Al3+ is 0.3 × 104 M-1. The limit of detection (LoD) for Al3+ and L-cysteine are 2.02 × 10-7 M and 0.5 × 10-5 M respectively. The probe (R) can show maximum efficiency within the pH range (7.0-10.0). The probe is found non-toxic (>80 % cell viability with 15 µM concentration) and employed for the in-vitro fluorescence imaging in the HeLa cell.

Nanoalum adjuvanted vaccines: small details make a big difference.

Purified vaccine antigens offer important safety and reactogenicity advantages compared with live attenuated or whole killed virus and bacterial vaccines. However, they require the addition of adjuvants to induce the magnitude, duration and quality of immune response required to achieve protective immunity. Aluminium salts have been used as adjuvants in vaccines for almost a century. In the literature, they are often referred to as aluminium-based adjuvants (ABAs), or aluminium salt-containing adjuvants or more simply "alum". All these terms are used to group aluminium suspensions that are very different in terms of atomic composition, size, and shape. They differ also in stability, antigen-adsorption, and antigen-release kinetics. Critically, these parameters also have a profound effect on the character and magnitude of the immune response elicited. Recent findings suggest that, by reducing the size of aluminium from micro to nanometers, a more effective adjuvant is obtained, together with the ability to sterile filter the vaccine product. However, the behaviour of aluminium nanoparticles in vaccine formulations is different from microparticles, requiring specific formulation strategies, as well as a more detailed understanding of how formulation influences the immune response generated. Here we review the current state of art of aluminium nanoparticles as adjuvants, with a focus on their immunobiology, preparation methods, formulation optimisation and stabilisation.

Boron supply restores aluminum-blocked auxin transport by the modulation of PIN2 trafficking in the root apical transition zone.

The supply of boron (B) alleviates the toxic effects of aluminum (Al) on root growth; however, the mechanistic basis of this process remains elusive. This study filled this knowledge gap, demonstrating that boron modifies auxin distribution and transport in Al-exposed Arabidopsis roots. In B-deprived roots, treatment with Al induced an increase in auxin content in the root apical meristem zone (MZ) and transition zone (TZ), whereas in the elongation zone (EZ) the auxin content was decreased beyond the level required for adequate growth. These distribution patterns are explained by the fact that basipetal auxin transport from the TZ to the EZ was disrupted by Al-inhibited PIN-FORMED 2 (PIN2) endocytosis. Experiments involving the modulation of protein biosynthesis by cycloheximide (CHX) and transcriptional regulation by cordycepin (COR) demonstrated that the Al-induced increase of PIN2 membrane proteins was dependent upon the inhibition of PIN2 endocytosis, rather than on the transcriptional regulation of the PIN2 gene. Experiments reporting on the profiling of Al3+ and PIN2 proteins revealed that the inhibition of endocytosis of PIN2 proteins was the result of Al-induced limitation of the fluidity of the plasma membrane. The supply of B mediated the turnover of PIN2 endosomes conjugated with indole-3-acetic acid (IAA), and thus restored the Al-induced inhibition of IAA transport through the TZ to the EZ. Overall, the reported results demonstrate that boron supply mediates PIN2 endosome-based auxin transport to alleviate Al toxicity in plant roots.

Involvement of SlSTOP1 regulated SlFDH expression in aluminum tolerance by reducing NAD+ to NADH in the tomato root apex.

Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria-localized FDH with Km values of 2.087 mm formate and 29.1 µm NAD+ . Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase-polymerase chain reaction. CRISPR/Cas9-generated SlFDH knockout lines were more sensitive to Al stress and formate than wild-type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD+ accumulated in response to Al stress. Co-expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual-luciferase reporter assay and electrophoretic mobility shift assay. The Al-induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato.

Low levels of Al stimulate the aboveground growth of Davidia involucrata saplings.

Davidia involucrata is a woody perennial and the only living species in the Genus Davidia. It is native to southern China where it holds cultural and scientific importance. However, D. involucrata is now an endangered species and its natural range includes low pH soils which are increasingly impacted by acid rain, nitrogen deposition and imbalanced nutrient cycling. The combination of these stresses also poses the additional risk of aluminum (Al) toxicity. Since the responses of D. involucrata to low pH and aluminum toxicity have not been investigated previously, a hydroponic experiment was conducted to examine the growth of one year old D. involucrata saplings after 50 d growth in a range of pH and Al conditions. Plant biomass, morphology, antioxidant enzyme activity, mineral concentrations and plant ecological strategy were compared at pH 5.8 and pH 4.0 without added Al (AlCl3) and in 0.1, 0.2 and 0.5 mM Al at pH 4.0. Our results showed that compared with pH 5.8, pH 4.0 (without added Al) not only inhibited root and shoot growth but also limited accumulation of nitrogen (N) and phosphorus (P) in leaves of D. involucrate. However, low Al concentrations (0.1 and 0.2 mM Al) at pH 4.0 partially restored the aboveground growth and leaf N concentrations, suggesting an alleviation of H+ toxicity by low Al concentrations. Compared with low Al concentrations, 0.5 mM Al treatment decreased plant growth and concentrations of N, P, and magnesium (Mg) in the leaves, which demonstrated the toxicity of high Al concentration. The results based on plant ecological strategy showed that D. involucrate decreased the competitiveness and favored its stress tolerance as pH changed from 5.8 to 4.0. Meanwhile, the competitiveness and stress tolerance of D. involucrata increased and decreased at low Al concentrations, respectively, and decreased and increased at high Al concentration, respectively. These trade-offs in ecological strategy were consistent with the responses of growth and antioxidant enzyme activity, reflecting a sensitive adaptation of D. involucrata to acid and Al stresses, which may aid in sustaining population dynamics. These findings are meaningful for understanding the population dynamics of D. involucrata in response to aluminum toxicity in acid soils.

OsAlR3 regulates aluminum tolerance through promoting the secretion of organic acids and the expression of antioxidant genes in rice.

In acidic soils, aluminum (Al) toxicity inhibits the growth and development of plant roots and affects nutrient and water absorption, leading to reduced yield and quality. Therefore, it is crucial to investigate and identify candidate genes for Al tolerance and elucidate their physiological and molecular mechanisms under Al stress. In this study, we identified a new gene OsAlR3 regulating Al tolerance, and analyzed its mechanism from physiological, transcriptional and metabolic levels. Compared with the WT, malondialdehyde (MDA) and hydrogen peroxide (H2O2) content were significantly increased, superoxide dismutase (SOD) activity and citric acid (CA) content were significantly decreased in the osalr3 mutant lines when exposed to Al stress. Under Al stress, the osalr3 exhibited decreased expression of antioxidant-related genes and lower organic acid content compared with WT. Integrated transcriptome and metabolome analysis showed the phenylpropanoid biosynthetic pathway plays an important role in OsAlR3-mediated Al tolerance. Exogenous CA and oxalic acid (OA) could increase total root length and enhance the antioxidant capacity in the mutant lines under Al stress. Conclusively, we found a new gene OsAlR3 that positively regulates Al tolerance by promoting the chelation of Al ions through the secretion of organic acids, and increasing the expression of antioxidant genes.

Neurotoxicity Profiling of Aluminum Salt-Based Nanoparticles as Adjuvants for Therapeutic Cancer Vaccine.

Therapeutic vaccines containing aluminum adjuvants have been widely used in the treatment of tumors due to their powerful immune-enhancing effects. However, the neurotoxicity of aluminum adjuvants with different physicochemical properties has not been completely elucidated. In this study, a library of engineered aluminum oxyhydroxide (EAO) and aluminum hydroxyphosphate (EAHP) nanoparticles was synthesized to determine their neurotoxicity in vitro. It was demonstrated that the surface charge of EAHPs and size of EAOs did not affect the cytotoxicity in N9, bEnd.3, and HT22 cells; however, soluble aluminum ions trigger the cytotoxicity in three different cell lines. Moreover, soluble aluminum ions induce apoptosis in N9 cells, and further mechanistic studies demonstrated that this apoptosis was mediated by mitochondrial reactive oxygen species generation and mitochondrial membrane potential loss. This study identifies the safety profile of aluminum-containing salts adjuvants in the nervous system during therapeutic vaccine use, and provides novel design strategies for their safer applications. SIGNIFICANCE STATEMENT: In this study, it was demonstrated that engineered aluminum oxyhydroxide and aluminum hydroxyphosphate nanoparticles did not induce cytotoxicity in N9, bEnd.3, and HT22 cells. In comparation, soluble aluminum ions triggered significant cytotoxicity in three different cell lines, indicating that the form in which aluminum is presenting may play a crucial role in its safety. Moreover, apoptosis induced by soluble aluminum ions was dependent on mitochondrial damage. This study confirms the safety of engineered aluminum adjuvants in vaccine formulations.

A Platinum-Aluminum Bimetallic Salen Complex for Pro-senescence Cancer Therapy.

Cell senescence is defined as irreversible cell cycle arrest, which can be triggered by telomere shortening or by various types of genotoxic stress. Induction of senescence is emerging as a new strategy for the treatment of cancer, especially when sequentially combined with a second senolytic drug capable of killing the resulting senescent cells, however severely suffering from the undesired off-target side effects from the senolytic drugs. Here, we prepare a bimetalic platinum-aluminum salen complex (Alumiplatin) for cancer therapy-a combination of pro-senesence chemotherapy with in situ senotherapy to avoid the side effects. The aluminum salen moiety, as a G-quadruplex stabilizer, enhances the salen's ability to induce cancer cell senescence and this phenotype is in turn sensitive to the cytotoxic activity of the monofunctional platinum moiety. It exhibits an excellent capability for inducing senescence, a potent cytotoxic activity against cancer cells both in vitro and in vivo, and an improved safety profile compared to cisplatin. Therefore, Alumiplatin may be a good candidate to be further developed into safe and effective anticancer agents. This novel combination of cell senescence inducers with genotoxic drugs revolutionizes the therapy options of designing multi-targeting anticancer agents to improve the efficacy of anticancer therapies.

Auxin acts upstream of nitric oxide to regulate cell wall xyloglucan and its aluminium-binding capacity in Arabidopsis thaliana.

MAIN CONCLUSION: Auxin acts upstream of NO through NOA and XXT5 pathways to regulate the binding capacity of the root cell wall to Al. In our previous study, we identified an unknown mechanism by which 1-naphthaleneacetic acid (NAA) decreased the fixation of aluminum (Al) in the cell wall. Here, we observed that external application of the nitric oxide (NO) donor S-nitrosoglutathion (GSNO) increased the inhibition of Al on root elongation. Further analysis indicated that GSNO could induce Al accumulation in the roots and root cell walls, which is consistent with lower xyloglucan content. In comparison to the Columbia-0 (Col-0) wild type (WT), endogenous NO-reduced mutants noa1 (NOA pathway) and nia1nia2 (NR pathway) were more resistant to Al, with lower root Al content, higher xyloglucan content, and more Al accumulation in the root cell walls. By contrast, the xxt5 mutant with reduced xyloglucan content exhibited an Al-sensitive phenotype. Interestingly, Al treatment increased the endogenous auxin and NO levels, and the auxin levels induced under Al stress further stimulated NO production. Auxin application reduced Al retention in hemicellulose and decreased the xyloglucan content, similar to the effects observed with GSNO. In yucca and aux1-7 mutants, exogenous application of NO resulted in responses similar to those of the WT, whereas exogenous auxin had little effect on the noa1 mutant under Al stress. In addition, as auxin had similar effects on the nia1nia2 mutant and the WT, exogenous auxin and NO had little effect on the xxt5 mutant under Al stress, further confirming that auxin acts upstream of NO through NOA and XXT5 pathways to regulate the binding capacity of the root cell wall to Al.

γ-Aminobutyric acid (GABA) priming alleviates acid-aluminum toxicity to roots of creeping bentgrass via enhancements in antioxidant defense and organic metabolites remodeling.

MAIN CONCLUSION: γ-Aminobutyric acid alleviates acid-aluminum toxicity to roots associated with enhanced antioxidant metabolism as well as accumulation and transportation of citric and malic acids. Aluminum (Al) toxicity has become the main limiting factor for crop growth and development in acidic soils and is further being aggravated worldwide due to continuous industrial pollution. The current study was designed to examine effects of GABA priming on alleviating acid-Al toxicity in terms of root growth, antioxidant defense, citrate and malate metabolisms, and extensive metabolites remodeling in roots under acidic conditions. Thirty-seven-day-old creeping bentgrass (Agrostis stolonifera) plants were used as test materials. Roots priming with or without 0.5 mM GABA for 3 days were cultivated in standard nutrient solution for 15 days as control or subjected to nutrient solution containing 5 mM AlCl3·6H2O for 15 days as acid-Al stress treatment. Roots were sampled for determinations of root characteristics, physiological and biochemical parameters, and metabolomics. GABA priming significantly alleviated acid-Al-induced root growth inhibition and oxidative damage, despite it promoted the accumulation of Al in roots. Analysis of metabolomics showed that GABA priming significantly increased accumulations of organic acids, amino acids, carbohydrates, and other metabolites in roots under acid-Al stress. In addition, GABA priming also significantly up-regulated key genes related to accumulation and transportation of malic and citric acids in roots under acid-Al stress. GABA-regulated metabolites participated in tricarboxylic acid cycle, GABA shunt, antioxidant defense system, and lipid metabolism, which played positive roles in reactive oxygen species scavenging, energy conversion, osmotic adjustment, and Al ion chelation in roots.

CcNFYB3-CcMATE35 and LncRNA CcLTCS-CcCS modules jointly regulate the efflux and synthesis of citrate to enhance aluminium tolerance in pigeon pea.

Aluminium (Al) toxicity decreases crop production in acid soils in general, but many crops have evolved complex mechanisms to resist it. However, our current understanding of how plants cope with Al stress and perform Al resistance is still at the initial stage. In this study, the citrate transporter CcMATE35 was identified to be involved in Al stress response. The release of citrate was increased substantially in CcMATE35 over-expression (OE) lines under Al stress, indicating enhanced Al resistance. It was demonstrated that transcription factor CcNFYB3 regulated the expression of CcMATE35, promoting the release of citrate from roots to increase Al resistance in pigeon pea. We also found that a Long noncoding RNA Targeting Citrate Synthase (CcLTCS) is involved in Al resistance in pigeon pea. Compared with controls, overexpression of CcLTCS elevated the expression level of the Citrate Synthase gene (CcCS), leading to increases in root citrate level and citrate release, which forms another module to regulate Al resistance in pigeon pea. Simultaneous overexpression of CcNFYB3 and CcLTCS further increased Al resistance. Taken together, these findings suggest that the two modules, CcNFYB3-CcMATE35 and CcLTCS-CcCS, jointly regulate the efflux and synthesis of citrate and may play an important role in enhancing the resistance of pigeon pea under Al stress.

Th2 and Th17-associated immunopathology following SARS-CoV-2 breakthrough infection in Spike-vaccinated ACE2-humanized mice.

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety.

Melatonin promotes Al3+ compartmentalization via H+ transport and ion gradients in Malus hupehensis.

Soil acidification in apple (Malus domestica) orchards results in the release of rhizotoxic aluminum ions (Al3+) into soil. Melatonin (MT) participates in plant responses to abiotic stress; however, its role in AlCl3 stress in apple remains unknown. In this study, root application of MT (1 µM) substantially alleviated AlCl3 stress (300 µM) in Pingyi Tiancha (Malus hupehensis), which was reflected by higher fresh and dry weight, increased photosynthetic capacity, and longer and more roots compared with plants that did not receive MT treatment. MT functioned mainly by regulating vacuolar H+/Al3+ exchange and maintaining H+ homeostasis in the cytoplasm under AlCl3 stress. Transcriptome deep sequencing analysis identified the transcription factor gene SENSITIVE TO PROTON RHIZOTOXICITY 1 (MdSTOP1) was induced by both AlCl3 and MT treatments. Overexpressing MdSTOP1 in apple increased AlCl3 tolerance by enhancing vacuolar H+/Al3+ exchange and H+ efflux to the apoplast. We identified 2 transporter genes, ALUMINUM SENSITIVE 3 (MdALS3) and SODIUM HYDROGEN EXCHANGER 2 (MdNHX2), as downstream targets of MdSTOP1. MdSTOP1 interacted with the transcription factor NAM ATAF and CUC 2 (MdNAC2) to induce MdALS3 expression, which reduced Al toxicity by transferring Al3+ from the cytoplasm to the vacuole. Furthermore, MdSTOP1 and MdNAC2 coregulated MdNHX2 expression to increase H+ efflux from the vacuole to the cytoplasm to promote Al3+ compartmentalization and maintain cation balance in the vacuole. Taken together, our findings reveal an MT-STOP1 + NAC2-NHX2/ALS3-vacuolar H+/Al3+ exchange model for the alleviation of AlCl3 stress in apple, laying a foundation for practical applications of MT in agriculture.

Chloroplast-targeted late embryogenesis abundant 1 increases alfalfa tolerance to drought and aluminum.

Late embryogenesis-abundant (LEA) proteins are important stress-response proteins that participate in protecting plants against abiotic stresses. Here, we investigated LEA group 3 protein MsLEA1, containing the typically disordered and α-helix structure, via overexpression and RNA interference (RNAi) approaches in alfalfa (Medicago sativa L.) under drought and aluminum (Al) stresses. MsLEA1 was highly expressed in leaves and localized in chloroplasts. Overexpressing MsLEA1 increased alfalfa tolerance to drought and Al stresses, but downregulating MsLEA1 decreased the tolerance. We observed a larger stomatal aperture and a lower water use efficiency in MsLEA1 RNAi lines compared with wild-type plants under drought stress. Photosynthetic rate, Rubisco activity, and superoxide dismutase (SOD) activity increased or decreased in MsLEA1-OE or MsLEA1-RNAi lines, respectively, under drought and Al stress. Copper/zinc SOD (Cu/Zn-SOD), iron SOD (Fe-SOD), and Rubisco large subunit proteins (Ms1770) were identified as binding partners of MsLEA1, which protected chloroplast structure and function under drought and Al stress. These results indicate that MsLEA1 recruits and protects its target proteins (SOD and Ms1770) and increases alfalfa tolerance against drought and Al stresses.

Low-acidity ALUMINUM-DEPENDENT MALATE TRANSPORTER4 genotype determines malate content in cultivated jujube.

Jujube (Ziziphus jujuba Mill.), the most economically important fruit tree in Rhamnaceae, was domesticated from sour jujube (Z. jujuba Mill. var. spinosa (Bunge) Hu ex H.F.Chow.). During domestication, fruit sweetness increased and acidity decreased. Reduction in organic acid content is crucial for the increase in sweetness of jujube fruit. In this study, the determination of malate content among 46 sour jujube and 35 cultivated jujube accessions revealed that malate content varied widely in sour jujube (0.90-13.31 mg g-1) but to a lesser extent in cultivated jujube (0.33-2.81 mg g-1). Transcriptome sequencing analysis showed that the expression level of Aluminum-Dependent Malate Transporter 4 (ZjALMT4) was substantially higher in sour jujube than in jujube. Correlation analysis of mRNA abundance and fruit malate content and transient gene overexpression showed that ZjALMT4 participates in malate accumulation. Further sequencing analyses revealed that three genotypes of the W-box in the promoter of ZjALMT4 in sour jujube associated with malate content were detected, and the genotype associated with low malate content was fixed in jujube. Yeast one-hybrid screening showed that ZjWRKY7 binds to the W-box region of the high-acidity genotype in sour jujube, whereas the binding ability was weakened in jujube. Transient dual-luciferase and overexpression analyses showed that ZjWRKY7 directly binds to the promoter of ZjALMT4, activating its transcription, and thereby promoting malate accumulation. These findings provide insights into the mechanism by which ZjALMT4 modulates malate accumulation in sour jujube and jujube. The results are of theoretical and practical importance for the exploitation and domestication of germplasm resources.

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.

Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al.

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe2+ or Al3+ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe2+ or Al3+ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe2+ and Al3+ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe2+ or Al3+ is not sufficient for arresting root growth in our experimental conditions.

Mutator-like transposable element 9A interacts with metacaspase 1 and modulates the incidence of Al-induced programmed cell death in peanut.

The toxicity of aluminum (Al) in acidic soil inhibits plant root development and reduces crop yields. In the plant response to Al toxicity, the initiation of programmed cell death (PCD) appears to be an important mechanism for the elimination of Al-damaged cells to ensure plant survival. In a previous study, the type I metacaspase AhMC1 was found to regulate the Al stress response and to be essential for Al-induced PCD. However, the mechanism by which AhMC1 is altered in the peanut response to Al stress remained unclear. Here, we show that a nuclear protein, mutator-like transposable element 9A (AhMULE9A), directly interacts with AhMC1 in vitro and in vivo. This interaction occurs in the nucleus in peanut and is weakened during Al stress. Furthermore, a conserved C2HC zinc finger domain of AhMULE9A (residues 735-751) was shown to be required for its interaction with AhMC1. Overexpression of AhMULE9A in Arabidopsis and peanut strongly inhibited root growth with a loss of root cell viability under Al treatment. Conversely, knock down of AhMULE9A in peanut significantly reduced Al uptake and Al inhibition of root growth, and alleviated the occurrence of typical hallmarks of Al-induced PCD. These findings provide novel insight into the regulation of Al-induced PCD.