A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate inositol hexaphosphate accumulation.

Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.

The LRR receptor-like kinase ALR1 is a plant aluminum ion sensor.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration.

Beyond iron-storage pool: functions of plant apoplastic iron during stress.

Iron (Fe) is an essential micronutrient for plants, and its storage in the apoplast represents an important Fe pool. Plants have developed various strategies to reutilize this apoplastic Fe pool to adapt to Fe deficiency. In addition, growing evidence indicates that the dynamic changes in apoplastic Fe are critical for plant adaptation to other stresses, including ammonium stress, phosphate deficiency, and pathogen attack. In this review, we discuss and scrutinize the relevance of apoplastic Fe for plant behavior changes in response to stress cues. We mainly focus on the relevant components that modulate the actions and downstream events of apoplastic Fe in stress signaling networks.

ART1 and putrescine contribute to rice aluminum resistance via OsMYB30 in cell wall modification.

Cell wall is the first physical barrier to aluminum (Al) toxicity. Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance; nevertheless, how it is regulated in rice remains largely unknown. In this study, we show that exogenous application of putrescines (Put) could significantly restore the Al resistance of art1, a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1 (ART1), and reduce its Al accumulation particularly in the cell wall of root tips. Based on RNA-sequencing, yeast-one-hybrid and electrophoresis mobility shift assays, we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance. Furthermore, transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30, and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation, hence reducing the Al-binding capacity of cell wall and enhancing Al resistance. Additionally, Put repressed OsMYB30 expression by eliminating Al-induced H2 O2 accumulation, while exogenous H2 O2 promoted OsMYB30 expression. We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice.

Ethylene signaling modulates Arabidopsis thaliana nitrate metabolism.

MAIN CONCLUSION: Genetic analysis reveals a previously unknown role for ethylene signaling in regulating Arabidopsis thaliana nitrogen metabolism. Nitrogen (N) is essential for plant growth, and assimilation of soil nitrate (NO3-) and ammonium ions is an important route of N acquisition. Although N import and assimilation are subject to multiple regulatory inputs, the extent to which ethylene signaling contributes to this regulation remains poorly understood. Here, our analysis of Arabidopsis thaliana ethylene signaling mutants advances that understanding. We show that the loss of CTR1 function ctr1-1 mutation confers resistance to the toxic effects of the NO3- analogue chlorate (ClO3-), and reduces the activity of the nitrate reductase (NR) enzyme of NO3- assimilation. Our further analysis indicates that the lack of the downstream EIN2 component (conferred by novel ein2 mutations) suppresses the effect of ctr1-1, restoring ClO3- sensitivity and NR activity to normal. Collectively, our observations indicate an important role for ethylene signaling in regulating Arabidopsis thaliana NO3- metabolism. We conclude that ethylene signaling enables environmentally responsive coordination of plant growth and N metabolism.

Phloem iron remodels root development in response to ammonium as the major nitrogen source.

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH4+ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

Structural basis of ALMT1-mediated aluminum resistance in Arabidopsis.

The plant aluminum (Al)-activated malate transporter ALMT1 mediates the efflux of malate to chelate the Al in acidic soils and underlies the plant Al resistance. Here we present cryo-electron microscopy (cryo-EM) structures of Arabidopsis thaliana ALMT1 (AtALMT1) in the apo, malate-bound, and Al-bound states at neutral and/or acidic pH at up to 3.0 Å resolution. The AtALMT1 dimer assembles an anion channel and each subunit contains six transmembrane helices (TMs) and six cytosolic α-helices. Two pairs of Arg residues are located in the center of the channel pore and contribute to malate recognition. Al binds at the extracellular side of AtALMT1 and induces conformational changes of the TM1-2 loop and the TM5-6 loop, resulting in the opening of the extracellular gate. These structures, along with electrophysiological measurements, molecular dynamic simulations, and mutagenesis study in Arabidopsis, elucidate the structural basis for Al-activated malate transport by ALMT1.

Restriction of iron loading into developing seeds by a YABBY transcription factor safeguards successful reproduction in Arabidopsis.

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression.

Ethylene promotes seed iron storage during Arabidopsis seed maturation via ERF95 transcription factor.

Because Iron (Fe) is an essential element, Fe storage in plant seeds is necessary for seedling establishment following germination. However, the mechanisms controlling seed Fe storage during seed development remain largely unknown. Here we reveal that an ERF95 transcription factor regulates Arabidopsis seed Fe accumulation. We show that expression of ERF95 increases during seed maturation, and that lack of ERF95 reduces seed Fe accumulation, consequently increasing sensitivity to Fe deficiency during seedling establishment. Conversely, overexpression of ERF95 has the opposite effects. We show that lack of ERF95 decreases abundance of FER1 messenger RNA in developing seed, which encodes Fe-sequestering ferritin. Accordingly, a fer1-1 loss-of-function mutation confers reduced seed Fe accumulation, and suppresses ERF95-promoted seed Fe accumulation. In addition, ERF95 binds to specific FER1 promoter GCC-boxes and transactivates FER1 expression. We show that ERF95 expression in maturing seed is dependent on EIN3, the master transcriptional regulator of ethylene signaling. While lack of EIN3 reduces seed Fe content, overexpression of ERF95 rescues Fe accumulation in the seed of ein3 loss-of-function mutant. Finally, we show that ethylene production increases during seed maturation. We conclude that ethylene promotes seed Fe accumulation during seed maturation via an EIN3-ERF95-FER1-dependent signaling pathway.

Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress.

BACKGROUND: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. RESULTS: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up- or down-regulated in roots of tomato following Al stress. CONCLUSION: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Jasmonic acid alleviates cadmium toxicity in Arabidopsis via suppression of cadmium uptake and translocation.

Jasmonic acid (JA) is thought to be involved in plant responses to cadmium (Cd) stress, but the underlying molecular mechanisms are poorly understood. Here, we show that Cd treatment rapidly induces the expression of genes promoting endogenous JA synthesis, and subsequently increases the JA concentration in Arabidopsis roots. Furthermore, exogenous methyl jasmonate (MeJA) alleviates Cd-generated chlorosis of new leaves by decreasing the Cd concentration in root cell sap and shoot, and decreasing the expression of the AtIRT1, AtHMA2 and AtHMA4 genes promoting Cd uptake and long-distance translocation, respectively. In contrast, mutation of a key JA synthesis gene, AtAOS, greatly enhances the expression of AtIRT1, AtHMA2 and AtHMA4, increases Cd concentration in both roots and shoots, and confers increased sensitivity to Cd. Exogenous MeJA recovers the enhanced Cd-sensitivity of the ataos mutant, but not of atcoi1, a JA receptor mutant. In addition, exogenous MeJA reduces NO levels in Cd-stressed Arabidopsis root tips. Taken together, our results suggest that Cd-induced JA acts via the JA signaling pathway and its effects on NO levels to positively restrict Cd accumulation and alleviates Cd toxicity in Arabidopsis via suppression of the expression of genes promoting Cd uptake and long-distance translocation.

A WRKY transcription factor confers aluminum tolerance via regulation of cell wall modifying genes.

Modification of cell wall properties has been considered as one of the determinants that confer aluminum (Al) tolerance in plants, while how cell wall modifying processes are regulated remains elusive. Here, we present a WRKY transcription factor WRKY47 involved in Al tolerance and root growth. Lack of WRKY47 significantly reduces, while overexpression of it increases Al tolerance. We show that lack of WRKY47 substantially affects subcellular Al distribution in the root, with Al content decreased in apoplast and increased in symplast, which is attributed to the reduced cell wall Al-binding capacity conferred by the decreased content of hemicellulose I in the wrky47-1 mutant. Based on microarray, real time-quantitative polymerase chain reaction and chromatin immunoprecipitation assays, we further show that WRKY47 directly regulates the expression of EXTENSIN-LIKE PROTEIN (ELP) and XYLOGLUCAN ENDOTRANSGLUCOSYLASE-HYDROLASES17 (XTH17) responsible for cell wall modification. Increasing the expression of ELP and XTH17 rescued Al tolerance as well as root growth in wrky47-1 mutant. In summary, our results demonstrate that WRKY47 is required for root growth under both normal and Al stress conditions via direct regulation of cell wall modification genes, and that the balance of Al distribution between root apoplast and symplast conferred by WRKY47 is important for Al tolerance.

Mechanisms and regulation of aluminum-induced secretion of organic acid anions from plant roots.

Aluminum (Al) is the most abundant metal element in the earth's crust. On acid soils, at pH 5.5 or lower, part of insoluble Al-containing minerals become solubilized into soil solution, with resultant highly toxic effects on plant growth and development. Nevertheless, some plants have developed Al-tolerance mechanisms that enable them to counteract this Al toxicity. One such well-documented mechanism is the Al-induced secretion of organic acid anions, including citrate, malate, and oxalate, from plant roots. Once secreted, these anions chelate external Al ions, thus protecting the secreting plant from Al toxicity. Genes encoding the citrate and malate transporters responsible for secretion have been identified and characterized, and accumulating evidence indicates that regulation of the expression of these transporter genes is critical for plant Al tolerance. In this review, we outline the recent history of research into plant Al-tolerance mechanisms, with special emphasis on the physiology of Al-induced secretion of organic acid anions from plant roots. In particular, we summarize the identification of genes encoding organic acid transporters and review current understanding of genes regulating organic acid secretion. We also discuss the possible signaling pathways regulating the expression of organic acid transporter genes.

A feedback loop between CaWRKY41 and H2O2 coordinates the response to Ralstonia solanacearum and excess cadmium in pepper.

WRKY transcription factors have been implicated in both plant immunity and plant responses to cadmium (Cd); however, the mechanism underlying the crosstalk between these processes is unclear. Here, we characterized the roles of CaWRKY41, a group III WRKY transcription factor, in immunity against the pathogenic bacterium Ralstonia solanacearum and Cd stress responses in pepper (Capsicum annuum). CaWRKY41 was transcriptionally up-regulated in response to Cd exposure, R. solanacearum inoculation, and H2O2 treatment. Virus-induced silencing of CaWRKY41 increased Cd tolerance and R. solanacearum susceptibility, while heterologous overexpression of CaWRKY41 in Arabidopsis impaired Cd tolerance, and enhanced Cd and zinc (Zn) uptake and H2O2 accumulation. Genes encoding reactive oxygen species-scavenging enzymes were down-regulated in CaWRKY41-overexpressing Arabidopsis plants, whereas genes encoding Zn transporters and enzymes involved in H2O2 production were up-regulated. Consistent with these findings, the ocp3 (overexpressor of cationic peroxidase 3) mutant, which has elevated H2O2 levels, displayed enhanced sensitivity to Cd stress. These results suggest that a positive feedback loop between H2O2 accumulation and CaWRKY41 up-regulation coordinates the responses of pepper to R. solanacearum inoculation and Cd exposure. This mechanism might reduce Cd tolerance by increasing Cd uptake via Zn transporters, while enhancing resistance to R. solanacearum.

Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa).

Whilst WRKY transcription factors are known to be involved in diverse plant responses to biotic stresses, their involvement in abiotic stress tolerance is poorly understood. OsFRDL4, encoding a citrate transporter, has been reported to be regulated by ALUMINUM (Al) RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) in rice, but whether it is also regulated by other transcription factors is unknown. We define the role of OsWRKY22 in response to Al stress in rice by using mutation and transgenic complementation assays, and characterize the regulation of OsFRDL4 by OsWRKY22 via yeas one-hybrid, electrophoretic mobility shift assay and ChIP-quantitative PCR. We demonstrate that loss of OsWRKY22 function conferred by the oswrky22 T-DNA insertion allele causes enhanced sensitivity to Al stress, and a reduction in Al-induced citrate secretion. We next show that OsWRKY22 is localized in the nucleus, functions as a transcriptional activator and is able to bind to the promoter of OsFRDL4 via W-box elements. Finally, we find that both OsFRDL4 expression and Al-induced citrate secretion are significantly lower in art1 oswrky22 double mutants than in the respective single mutants. We conclude that OsWRKY22 promotes Al-induced increases in OsFRDL4 expression, thus enhancing Al-induced citrate secretion and Al tolerance in rice.

Two citrate transporters coordinately regulate citrate secretion from rice bean root tip under aluminum stress.

Aluminum (Al)-induced organic acid secretion from the root apex is an important Al resistance mechanism. However, it remains unclear how plants fine-tune root organic acid secretion which can contribute significantly to the loss of fixed carbon from the plant. Here, we demonstrate that Al-induced citrate secretion from the rice bean root apex is biphasic, consisting of an early phase with low secretion and a later phase of large citrate secretion. We isolated and characterized VuMATE2 as a possible second citrate transporter in rice bean functioning in tandem with VuMATE1, which we previously identified. The time-dependent kinetics of VuMATE2 expression correlates well with the kinetics of early phase root citrate secretion. Ectopic expression of VuMATE2 in Arabidopsis resulted in increased root citrate secretion and Al resistance. Electrophysiological analysis of Xenopus oocytes expressing VuMATE2 indicated VuMATE2 mediates anion efflux. However, the expression regulation of VuMATE2 differs from VuMATE1. While a protein translation inhibitor suppressed Al-induced VuMATE1 expression, it releases VuMATE2 expression. Yeast one-hybrid assays demonstrated that a previously identified transcription factor, VuSTOP1, interacts with the VuMATE2 promoter at a GGGAGG cis-acting motif. Thus, we demonstrate that plants adapt to Al toxicity by fine-tuning root citrate secretion with two separate root citrate transport systems.

DNA mismatch repair preferentially protects genes from mutation.

Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth.

An Oxalyl-CoA Synthetase Is Involved in Oxalate Degradation and Aluminum Tolerance.

Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with Km = 121 ± 8.2 µm and Vmax of 7.7 ± 0.88 µmol min-1 mg-1 protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wild-type plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms.

Ethylene is involved in root phosphorus remobilization in rice (Oryza sativa) by regulating cell-wall pectin and enhancing phosphate translocation to shoots.

Background and aims Plants are able to grow under phosphorus (P)-deficient conditions by coordinating Pi acquisition, translocation from roots to shoots and remobilization within the plant. Previous reports have demonstrated that cell-wall pectin contributes greatly to rice cell-wall Pi re-utilization under P-deficient conditions, but whether other factors such as ethylene also affect the pectin-remobilizing capacity remains unclear. Methods Two rice cultivars, 'Nipponbare' (Nip) and 'Kasalath' (Kas) were cultured in the +P (complete nutrient solution), -P (withdrawing P from the complete nutrient solution), +P+ACC (1-amino-cyclopropane-1-carboxylic acid, an ethylene precursor, adding 1 µm ACC to the complete nutrient solution) and -P+ACC (adding 1 µm ACC to -P nutrient solution) nutrient solutions for 7 d. Key Results After 7 d -P treatment, there was clearly more soluble P in Nip root and shoot, accompanied by additional production of ethylene in Nip root compared with Kas. Under P-deficient conditions, addition of ACC significantly increased the cell-wall pectin content and decreased cell-wall retained P, and thus more soluble P was released to the root and translocated to the shoot, which was mediated by the expression of the P deficiency-responsive gene OsPT2, which also strongly induced by ACC treatment under both P-sufficient and P-deficient conditions. Conclusions Ethylene positively regulates pectin content and expression of OsPT2, which ultimately makes more P available by facilitating the solubilization of P fixed in the cell wall and its translocation to the shoot.

A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH.

Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H(+)) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H(+) stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H(+) stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H(+)- and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H(+) stress due to less production of formate in the transgenic tobacco lines under Al and H(+) stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H(+) stress coexist.