Transcription factor module NLP-NIGT1 fine-tunes NITRATE TRANSPORTER2.1 expression.

Arabidopsis (Arabidopsis thaliana) high-affinity NITRATE TRANSPORTER2.1 (NRT2.1) plays a dominant role in the uptake of nitrate, the most important nitrogen (N) source for most terrestrial plants. The nitrate-inducible expression of NRT2.1 is regulated by NIN-LIKE PROTEIN (NLP) family transcriptional activators and NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) family transcriptional repressors. Phosphorus (P) availability also affects the expression of NRT2.1 because the PHOSPHATE STARVATION RESPONSE1 transcriptional activator activates NIGT1 genes in P-deficient environments. Here, we show a biology-based mathematical understanding of the complex regulation of NRT2.1 expression by multiple transcription factors using 2 different approaches: a microplate-based assay for the real-time measurement of temporal changes in NRT2.1 promoter activity under different nutritional conditions, and an ordinary differential equation (ODE)-based mathematical modeling of the NLP- and NIGT1-regulated expression patterns of NRT2.1. Both approaches consistently reveal that NIGT1 stabilizes the amplitude of NRT2.1 expression under a wide range of nitrate concentrations. Furthermore, the ODE model suggests that parameters such as the synthesis rate of NIGT1 mRNA and NIGT1 proteins and the affinity of NIGT1 proteins for the NRT2.1 promoter substantially influence the temporal expression patterns of NRT2.1 in response to nitrate. These results suggest that the NLP-NIGT1 feedforward loop allows a precise control of nitrate uptake. Hence, this study paves the way for understanding the complex regulation of nutrient acquisition in plants, thus facilitating engineered nutrient uptake and plant response patterns using synthetic biology approaches.

NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis.

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined.

Below-ground plant-soil interactions affecting adaptations of rice to iron toxicity.

Iron toxicity is a major constraint to rice production, particularly in highly weathered soils of inland valleys in sub-Saharan Africa where the rice growing area is rapidly expanding. There is a wide variation in tolerance of iron toxicity in the rice germplasm. However, the introgression of tolerance traits into high-yielding germplasm has been slow owing to the complexity of the tolerance mechanisms and large genotype-by-environment effects. We review current understanding of tolerance mechanisms, particularly those involving below-ground plant-soil interactions. Until now these have been less studied than above-ground mechanisms. We cover processes in the rhizosphere linked to exclusion of toxic ferrous iron by oxidation, and resulting effects on the mobility of nutrient ions. We also cover the molecular physiology of below-ground processes controlling iron retention in roots and root-shoot transport, and also plant iron sensing. We conclude that future breeding programmes should be based on well-characterized molecular markers for iron toxicity tolerance traits. To successfully identify such markers, the complex tolerance response should be broken down into its components based on understanding of tolerance mechanisms, and tailored screening methods should be developed for individual mechanisms.

Nitrate-inducible NIGT1 proteins modulate phosphate uptake and starvation signalling via transcriptional regulation of SPX genes.

Nitrogen and phosphorus are two major soil nutrients required for plant growth. Because requirements of both these elements are interdependent, acquisition of one must be balanced with that of the other. However, the mechanism underlying this balanced acquisition remains unclear. Here, we show by in vivo luciferase imaging that the presence of nitrogen sources is a pre-requisite for strong activation of phosphate starvation responses. In addition, we also show that nitrate rather than ammonium is a potent modulator of phosphate starvation-induced gene expression. Furthermore, protoplast-based transient expression assay and chromatin immunoprecipitation assay demonstrate that NIGT1 GARP-type transcriptional repressors, which are encoded by nitrate-inducible genes, directly bind to and repress the promoters of genes encoding SPX proteins. Consistent with the role of SPX proteins in the suppression of the PHR1 transcriptional activator, the master regulator for phosphate starvation responses, nitrate-dependent enhancement of phosphate starvation responses, such as accumulation of anthocyanin and promotion of root hair growth and phosphate uptake, was less evident in the nigt1.1-nigt1.4 quadruple mutant. Consistently, NIGT1 overexpression alleviated the reduction in phosphate uptake under phosphate-replete conditions. We further reveal the intricate feedback regulations involving PHR1, NIGT1, and SPX family proteins in the phosphate starvation signalling network. Importantly, results of mutant protoplast-based assays and in planta analysis using NIGT1 overexpression in the spx1 spx2 double mutant indicated that the NIGT1-SPX-PHR cascade mediates nitrogen status-responsive regulation of phosphate uptake and starvation signalling. These findings uncover the mechanism underlying the balanced acquisition of nitrogen and phosphorus.

Environmental Control of Phosphorus Acquisition: A Piece of the Molecular Framework Underlying Nutritional Homeostasis.

Homeostasis of phosphorus (P), an essential macronutrient, is vital for plant growth under diverse environmental conditions. Although plants acquire P from the soil as inorganic phosphate (Pi), its availability is generally limited. Therefore, plants employ mechanisms involving various Pi transporters that facilitate efficient Pi uptake against a steep concentration gradient across the plant-soil interface. Among the different types of Pi transporters in plants, some members of the PHOSPHATE TRANSPORTER 1 (PHT1) family, present in the plasma membrane of root epidermal cells and root hairs, are chiefly responsible for Pi uptake from the rhizosphere. Therefore, accurate regulation of PHT1 expression is crucial for the maintenance of P homeostasis. Previous investigations positioned the Pi-dependent posttranslational regulation of PHOSPHATE STARVATION RESPONSE 1 (PHR1) transcription factor activity at the center of the regulatory mechanism controlling PHT1 expression and P homeostasis; however, recent studies indicate that several other factors also regulate the expression of PHT1 to modulate P acquisition and sustain P homeostasis against environmental fluctuations. Together with PHR1, several transcription factors that mediate the availability of other nutrients (such as nitrogen and zinc), light, and stress signals form an intricate transcriptional network to maintain P homeostasis under highly diverse environments. In this review, we summarize this intricate transcriptional network for the maintenance of P homeostasis under different environmental conditions, with a main focus on the mechanisms identified in Arabidopsis.

Gene regulatory network and its constituent transcription factors that control nitrogen-deficiency responses in rice.

Increase in the nitrogen (N)-use efficiency and optimization of N response in crop species are urgently needed. Although transcription factor-based genetic engineering is a promising approach for achieving these goals, transcription factors that play key roles in the response to N deficiency have not been studied extensively. Here, we performed RNA-seq analysis of root samples of 20 Asian rice (Oryza sativa) accessions with differential nutrient uptake. Data obtained from plants exposed to N-replete and N-deficient conditions were subjected to coexpression analysis and machine learning-based pathway inference to dissect the gene regulatory network required for the response to N deficiency. Four transcription factors, including members of the G2-like and bZIP families, were predicted to function as key regulators of gene transcription within the network in response to N deficiency. Cotransfection assays validated inferred novel regulatory pathways, and further analyses using genome-edited knockout lines suggested that these transcription factors are important for N-deficiency responses in planta. Many of the N deficiency-responsive genes, including those encoding key regulators within the network, were coordinately regulated by transcription factors belonging to different families. Transcription factors identified in this study could be valuable for the modification of N response and metabolism.

Metabolomic markers and physiological adaptations for high phosphate utilization efficiency in rice.

Utilizing phosphate more efficiently is crucial for sustainable crop production. Highly efficient rice (Oryza sativa) cultivars have been identified and this study aims to identify metabolic markers associated with P utilization efficiency (PUE). P deficiency generally reduced leaf P concentrations and CO2 assimilation rates but efficient cultivars were reducing leaf P concentrations further than inefficient ones while maintaining similar CO2 assimilation rates. Adaptive changes in carbon metabolism were detected but equally in efficient and inefficient cultivar groups. Groups furthermore did not differ with respect to partial substitutions of phospholipids by sulfo- and galactolipids. Metabolites significantly more abundant in the efficient group, such as sinapate, benzoate and glucoronate, were related to antioxidant defence and may help alleviating oxidative stress caused by P deficiency. Sugar alcohols ribitol and threitol were another marker metabolite for higher phosphate efficiency as were several amino acids, especially threonine. Since these metabolites are not known to be associated with P deficiency, they may provide novel clues for the selection of more P efficient genotypes. In conclusion, metabolite signatures detected here were not related to phosphate metabolism but rather helped P efficient lines to keep vital processes functional under the adverse conditions of P starvation.

Perception, transduction, and integration of nitrogen and phosphorus nutritional signals in the transcriptional regulatory network in plants.

Nitrate and phosphate ions are major sources of nitrogen and phosphorus for plants. In addition to their vital roles as indispensable macronutrients, these ions function as signalling molecules and induce a variety of responses. Plants adapt to different levels of nutrients by altering their gene expression profile and subsequent physiological and morphological responses. Advances made in recent years have provided novel insights into plant nutrient sensing and modulation of gene expression. Key breakthroughs include elucidation of the mechanisms underlying post-translational regulation of NIN-LIKE PROTEIN (NLP) and PHOSPHATE STARVATION RESPONSE (PHR) family transcription factors, which function as master regulators of responses to nitrate and phosphate starvation, respectively. Determination of the mechanisms whereby these nutrient signals are integrated through NIGT1/HHO family proteins has likewise represented important progress. Further studies have revealed novel roles in nutrient signalling of transcription factors that have previously been shown to be associated with other signals, such as light and phytohormones. Nitrate and phosphate signals are thus transmitted through an intricate gene regulatory network with the help of various positive and negative transcriptional regulators. These complex regulatory patterns enable plants to integrate input signals from various environmental factors and trigger appropriate responses, as exemplified by the regulatory module involving NIGT1/HHO family proteins. These mechanisms collectively support nutrient homeostasis in plants.

Shoot tolerance mechanisms to iron toxicity in rice (Oryza sativa L.).

Iron toxicity frequently affects lowland rice and leads to oxidative stress via the Fenton reaction. Tolerance mechanisms were investigated in contrasting genotypes: the intolerant IR29 and the tolerant recombinant inbred line FL483. Seedlings were exposed to 1000 mg L-1 ferrous iron, and the regulation of genes involved in three hypothetical tolerance mechanisms was investigated (I) Iron uptake, partitioning and storage. The iron concentration and speciation in different plant tissues did not differ significantly between genotypes. Sub-cellular iron partitioning genes such as vacuolar iron transporters or ferritin showed no genotypic differences. (II) Antioxidant biosynthesis. Only one gene involved in carotenoid biosynthesis showed genotypic differences, but carotenoids are unlikely to scavenge the reactive oxygen species (ROS) involved in Fe toxicity, i.e. H2 O2 and hydroxyl radicals. (III) Enzymatic activities for ROS scavenging and antioxidants turnover. In shoots, glutathione-S-transferase and ascorbate oxidase genes showed genotypic differences, and consistently, the tolerant FL483 had lower dehydroascorbate reductase and higher ascorbate oxidase activity, suggesting that high rates ascorbate reduction confer sensitivity. This hypothesis was confirmed by application of exogenous reduced ascorbate or L-galactono-1,4-lactone, which increased lipid peroxidation under iron toxic conditions. Our results demonstrate in planta pro-oxidant activity of reduced ascorbate in the presence of iron.

A Novel Gene, OZONE-RESPONSIVE APOPLASTIC PROTEIN1, Enhances Cell Death in Ozone Stress in Rice.

A novel protein, OZONE-RESPONSIVE APOPLASTIC PROTEIN1 (OsORAP1), was characterized, which was previously suggested as a candidate gene underlying OzT9, a quantitative trait locus for ozone stress tolerance in rice (Oryza sativa). The sequence of OsORAP1 was similar to that of ASCORBATE OXIDASE (AO) proteins. It was localized in the apoplast, as shown by transient expression of an OsORAP1/green fluorescent protein fusion construct in Nicotiana benthamiana leaf epidermal and mesophyll cells, but did not possess AO activity, as shown by heterologous expression of OsORAP1 in Arabidopsis (Arabidopsis thaliana) mutants with reduced background AO activity. A knockout rice line of OsORAP1 showed enhanced tolerance to ozone stress (120 nL L(-1) average daytime concentration, 20 d), as demonstrated by less formation of leaf visible symptoms (i.e. cell death), less lipid peroxidation, and lower NADPH oxidase activity, indicating reduced active production of reactive oxygen species. In contrast, the effect of ozone on chlorophyll content was not significantly different among the lines. These observations suggested that OsORAP1 specifically induced cell death in ozone stress. Significantly enhanced expression of jasmonic acid-responsive genes in the knockout line implied the involvement of the jasmonic acid pathway in symptom mitigation. Sequence analysis revealed extensive polymorphisms in the promoter region of OsORAP1 between the ozone-susceptible cv Nipponbare and the ozone-tolerant cv Kasalath, the OzT9 donor variety, which could be responsible for the differential regulation of OsORAP1 reported earlier. These pieces of evidence suggested that OsORAP1 enhanced cell death in ozone stress, and its expression levels could explain the effect of a previously reported quantitative trait locus.

Ascorbate biosynthesis and its involvement in stress tolerance and plant development in rice (Oryza sativa L.).

Ascorbic acid (AsA) biosynthesis and its implications for stress tolerance and plant development were investigated in a set of rice knock-out (KO) mutants for AsA biosynthetic genes and their wild-types. KO of two isoforms of GDP-D-mannose epimerase (OsGME) reduced the foliar AsA level by 20-30%, and KO of GDP-L-galactose phosphorylase (OsGGP) by 80%, while KO of myo-inositol oxygenase (OsMIOX) did not affect foliar AsA levels. AsA concentration was negatively correlated with lipid peroxidation in foliar tissue under ozone stress and zinc deficiency, but did not affect the sensitivity to iron toxicity. Lack of AsA reduced the photosynthetic efficiency as represented by the maximum carboxylation rate of Rubisco (Vmax), the maximum electron transport rate (Jmax) and the chlorophyll fluorescence parameter ΦPSII. Mutants showed lower biomass production than their wild-types, especially when OsGGP was lacking (around 80% reductions). All plants except for KO mutants of OsGGP showed distinct peaks in foliar AsA concentrations during the growth, which were consistent with up-regulation of OsGGP, suggesting that OsGGP plays a pivotal role in regulating foliar AsA levels during different growth stages. In conclusion, our data demonstrate multiple roles of AsA in stress tolerance and development of rice.

Genetic dissection of ozone tolerance in rice (Oryza sativa L.) by a genome-wide association study.

Tropospheric ozone causes various negative effects on plants and affects the yield and quality of agricultural crops. Here, we report a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with ozone tolerance. A diversity panel consisting of 328 accessions representing all subgroups of O. sativa was exposed to ozone stress at 60 nl l(-1) for 7h every day throughout the growth season, or to control conditions. Averaged over all genotypes, ozone significantly affected biomass-related traits (plant height -1.0%, shoot dry weight -15.9%, tiller number -8.3%, grain weight -9.3%, total panicle weight -19.7%, single panicle weight -5.5%) and biochemical/physiological traits (symptom formation, SPAD value -4.4%, foliar lignin content +3.4%). A wide range of genotypic variance in response to ozone stress were observed in all phenotypes. Association mapping based on more than 30 000 single-nucleotide polymorphism (SNP) markers yielded 16 significant markers throughout the genome by applying a significance threshold of P<0.0001. Furthermore, by determining linkage disequilibrium blocks associated with significant SNPs, we gained a total of 195 candidate genes for these traits. The following sequence analysis revealed a number of novel polymorphisms in two candidate genes for the formation of visible leaf symptoms, a RING and an EREBP gene, both of which are involved in cell death and stress defence reactions. This study demonstrated substantial natural variation of responses to ozone in rice and the possibility of using GWAS in elucidating the genetic factors underlying ozone tolerance.

Loci, genes, and mechanisms associated with tolerance to ferrous iron toxicity in rice (Oryza sativa L.).

KEY MESSAGE: A genome-wide association study in rice yielded loci and candidate genes associated with tolerance to iron toxicity, and revealed biochemical mechanisms associated with tolerance in contrasting haplotypes. Iron toxicity is a major nutrient disorder affecting rice. Therefore, understanding the genetic and physiological mechanisms associated with iron toxicity tolerance is crucial in adaptive breeding and biofortification. We conducted a genome-wide association study (GWAS) by exposing a population of 329 accessions representing all subgroups of rice to ferrous iron stress (1000 ppm, 5 days). Expression patterns and sequence polymorphisms of candidate genes were investigated, and physiological hypotheses related to candidate loci were tested using a subset of contrasting haplotypes. Both iron including and excluding tolerant genotypes were observed, and shoot iron concentrations explained around 15.5 % of the variation in foliar symptom formation. GWAS for seven traits yielded 20 SNP markers exceeding a significance threshold of -log10 P > 4.0, which represented 18 distinct loci. One locus mapped for foliar symptom formation on chromosome 1 contained two putative glutathione-S-transferases, which were strongly expressed under iron stress and showed sequence polymorphisms in complete linkage disequilibrium with the most significant SNP. Contrasting haplotypes for this locus showed significant differences in dehydroascorbate reductase activity, which affected the plants' redox status under iron stress. We conclude that maintaining foliar redox homeostasis under iron stress represented an important tolerance mechanism associated with a locus identified through GWAS.

[Mediastinal lymph node carcinoma of unknown primary site; report of a case].

We experienced a rare case of a 38-year-old man with mediastinal lymph node carcinoma of unknown primary site. He had an abnormal shadow of upper mediastinal mass in a health check, and he was referred to our hospital. Chest computed tomography( CT) showed only one swollen mediastinal lymph node, and positron emission tomography(PET) revealed an accumulation of fluorodeoxyglucose in the nodule. Serum levels of neuron specific enolase (NSE) were found to be elevated. Preoperative examination did not detect the primary lesion. She underwent a mediastinal lymphadenectomy. The pathological diagnosis of the lesion was large cell neuroendocrine carcinoma. He was given platinum-based combined chemotherapy and radiotherapy as adjuvant therapy. He has been well without any sign of recurrence for 1 year after surgery. Surgical resection of mediastinal lymph node carcinoma of unknown primary site has the possibility of a good prognosis.

[Giant hemangioma of the rib].

We experienced a rare case of a 73-year-old woman with giant hemangioma of the left 8th rib. She had an abnormal shadow of left lower lung field in a health check and was referred to our hospital. Preoperative examination suggested malignant rib tumor because of tumor growth beyond the disrupted bony cortex. She underwent resection of the left 8th rib with the 7th rib and the 7th intercostal muscles and reconstruction of the chest wall defect. The pathological diagnosis of the lesion was cavernous hemangioma. She was discharged after an uneventful postoperative course. She has been well without any sign of recurrence for 3 year after surgery.

[Pulmonary mycobacterium intracellulare infection complicated with pneumothorax and chronic empyema].

A 75-year-old woman who had been treated for pulmonary Mycobacterium intracellulare infection was admitted to a nearby hospital because of hemoptysis, right pneumothorax, and empyema. She had been treated by thoracic drainage and pleural lavage, but was reffered to our hospital because of refractory empyema. Her chest radiograph and chest computed tomography( CT) showed right chronic empyema of which pleural aspirate was smear positive for acid-fast bacilli and positive for the polymerase chain reaction method(PCR)-Mycobacterium intracellulare. Serum levels of white blood cell and C-reactive protein(CRP) were found to be slightly elevated. She was treated with combined use of ethambutol, rifampicin, clarithromycin, and kanamycin and with pleural curettage by thoracoscopic surgery. After surgery additional treatment was done using urokinase which was administered into the thoracic cavity via an thoracic tube. Chronic empyema gradually improved with the treatment and the pleural effusion became bacterial free, enabling the patient to discharge from hospital without thoracic drainage.

In-frame mutation in rice TEOSINTE BRANCHED1 (OsTB1) improves productivity under phosphorus deficiency.

Tillering is an important trait in rice productivity. We introduced mutations into the coding region of rice TEOSINTE BRANCHED1 (OsTB1), which is a negative regulator of tillering, using CRISPR/Cas9. The frameshift mutants exhibited substantially enhanced tillering and produced 3.5 times more panicles than the non-mutated plants at maturity. This enhanced tillering resulted in increased spikelet number; however, grain yields did not increase due to substantially reduced filled grain rate and 1,000-grain weight. In contrast, in-frame mutations in OsTB1 had the effect of slightly increasing tiller numbers, and the in-frame mutants had 40% more panicles than non-mutated plants. The grain yield of in-frame mutants also did not increase on nutrient-rich soil; however, under phosphorus-deficient conditions, where tillering is constrained, the in-frame mutants gave a significantly higher grain yield than non-mutated plants due to higher spikelet number and maintained filled grain rate. Rice grassy tiller1 (OsGT1)/OsHox12, which is directly regulated by OsTB1 to suppress tillering, was moderately down-regulated in in-frame mutants, suggesting that OsTB1 with the in-frame mutation shows partial function of intact OsTB1 in regulating OsGT1/OsHox12. We propose that mildly enhanced tillering by in-frame mutation of OsTB1 can improve grain yield under low phosphorus conditions.

Novel QTL for Lateral Root Density and Length Improve Phosphorus Uptake in Rice (Oryza sativa L.).

The rice root system consists of two types of lateral roots, indeterminate larger L-types capable of further branching, and determinate, short, unbranched S-types. L-type laterals correspond to the typical lateral roots of cereals whereas S-type laterals are unique to rice. Both types contribute to nutrient and water uptake and genotypic variation for density and length of these laterals could be exploited in rice improvement to enhance adaptations to nutrient and water-limited environments. Our objectives were to determine how best to screen for lateral root density and length and to identify markers linked to genotypic variation for these traits. Using different growing media showed that screening in nutrient solution exposed genotypic variation for S-type and L-type density, but only the lateral roots of soil-grown plants varied for their lengths. A QTL mapping population developed from parents contrasting for lateral root traits was grown in a low-P field, roots were sampled, scanned and density and length of lateral roots measured. One QTL each was detected for L-type density (LDC), S-type density on crown root (SDC), S-type density on L-type (SDL), S-type length on L-type (SLL), and crown root number (RNO). The QTL for LDC on chromosome 5 had a major effect, accounting for 46% of the phenotypic variation. This strong positive effect was confirmed in additional field experiments, showing that lines with the donor parent allele at qLDC5 had 50% higher LDC. Investigating the contribution of lateral root traits to P uptake using stepwise regressions indicated LDC and RNO were most influential, followed by SDL. Simulating effects of root trait differences conferred by the main QTL in a P uptake model confirmed that qLDC5 was most effective in improving P uptake followed by qRNO9 for RNO and qSDL9 for S-type lateral density on L-type laterals. Pyramiding qLDC5 with qRNO9 and qSDL9 would be possible given that trade-offs between traits were not detected. Phenotypic selection for the RNO trait during variety development would be feasible, however, the costs of doing so reliably for lateral root density traits is prohibitive and markers identified here therefore provide the first opportunity to incorporate such traits into a breeding program.

Magnesium supply alleviates iron toxicity-induced leaf bronzing in rice through exclusion and tissue-tolerance mechanisms.

Introduction: Iron (Fe) toxicity is a widespread nutritional disorder in lowland rice causing growth retardation and leaf symptoms referred to as leaf bronzing. It is partly caused by an imbalance of nutrients other than Fe and supply of these is known to mitigate the toxicity. But the physiological and molecular mechanisms involved are unknown. Methods: We investigated the effect of magnesium (Mg) on Fe toxicity tolerance in a field study in the Central Highlands of Madagascar and in hydroponic experiments with excess Fe (300 mg Fe L-1). An RNA-seq analysis was conducted in a hydroponic experiment to elucidate possible mechanisms underlying Mg effects. Results and discussion: Addition of Mg consistently decreased leaf bronzing under both field and hydroponic conditions, whereas potassium (K) addition caused minor effects. Plants treated with Mg tended to have smaller shoot Fe concentrations in the field, suggesting enhanced exclusion at the whole-plant level. However, analysis of multiple genotypes showed that Fe toxicity symptoms were also mitigated without a concomitant decrease of Fe concentration, suggesting that increased Mg supply confers tolerance at the tissue level. The hydroponic experiments also suggested that Mg mitigated leaf bronzing without significantly decreasing Fe concentration or oxidative stress as assessed by the content of malondialdehyde, a biomarker for oxidative stress. An RNA-seq analysis revealed that Mg induced more changes in leaves than roots. Subsequent cis-element analysis suggested that NAC transcription factor binding sites were enriched in genes induced by Fe toxicity in leaves. Addition of Mg caused non-significant enrichment of the same binding sites, suggesting that NAC family proteins may mediate the effect of Mg. This study provides clues for mitigating Fe toxicity-induced leaf bronzing in rice.