Immuno-PET and Targeted α-Therapy Using Anti-Glypican-1 Antibody Labeled with 89Zr or 211At: A Theranostic Approach for Pancreatic Ductal Adenocarcinoma.

Glypican-1 (GPC1) is overexpressed in several solid cancers and is associated with tumor progression, whereas its expression is low in normal tissues. This study aimed to evaluate the potential of an anti-GPC1 monoclonal antibody (GPC1 mAb) labeled with 89Zr or 211At as a theranostic target in pancreatic ductal adenocarcinoma. Methods: GPC1 mAb clone 01a033 was labeled with 89Zr or 211At with a deferoxamine or decaborane linker, respectively. The internalization ability of GPC1 mAb was evaluated by fluorescence conjugation using a confocal microscope. PANC-1 xenograft mice (n = 6) were intravenously administered [89Zr]GPC1 mAb (0.91 ± 0.10 MBq), and PET/CT scanning was performed for 7 d. Uptake specificity was confirmed through a comparative study using GPC1-positive (BxPC-3) and GPC1-negative (BxPC-3 GPC1-knockout) xenografts (each n = 3) and a blocking study. DNA double-strand breaks were evaluated using the γH2AX antibody. The antitumor effect was evaluated by administering [211At]GPC1 mAb (∼100 kBq) to PANC-1 xenograft mice (n = 10). Results: GPC1 mAb clone 01a033 showed increased internalization ratios over time. One day after administration, a high accumulation of [89Zr]GPC1 mAb was observed in the PANC-1 xenograft (SUVmax, 3.85 ± 0.10), which gradually decreased until day 7 (SUVmax, 2.16 ± 0.30). The uptake in the BxPC-3 xenograft was significantly higher than in the BxPC-3 GPC1-knockout xenograft (SUVmax, 4.66 ± 0.40 and 2.36 ± 0.36, respectively; P = 0.05). The uptake was significantly inhibited in the blocking group compared with the nonblocking group (percentage injected dose per gram, 7.3 ± 1.3 and 12.4 ± 3.0, respectively; P = 0.05). DNA double-strand breaks were observed by adding 150 kBq of [211At]GPC1 and were significantly suppressed by the internalization inhibitor (dynasore), suggesting a substantial contribution of the internalization ability to the antitumor effect. Tumor growth suppression was observed in PANC-1 mice after the administration of [211At]GPC1 mAb. Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [211At]GPC1 mAb, suggesting an essential role in targeted α-therapy. Conclusion: [89Zr]GPC1 mAb PET showed high tumoral uptake in the early phase after administration, and targeted α-therapy using [211At]GPC1 mAb showed tumor growth suppression. GPC1 is a promising target for future applications for the precise diagnosis of pancreatic ductal adenocarcinoma and GPC1-targeted theranostics.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Development of a chub mackerel with less-aggressive fry stage by genome editing of arginine vasotocin receptor V1a2.

Genome editing is a technology that can remarkably accelerate crop and animal breeding via artificial induction of desired traits with high accuracy. This study aimed to develop a chub mackerel variety with reduced aggression using an experimental system that enables efficient egg collection and genome editing. Sexual maturation and control of spawning season and time were technologically facilitated by controlling the photoperiod and water temperature of the rearing tank. In addition, appropriate low-temperature treatment conditions for delaying cleavage, shape of the glass capillary, and injection site were examined in detail in order to develop an efficient and robust microinjection system for the study. An arginine vasotocin receptor V1a2 (V1a2) knockout (KO) strain of chub mackerel was developed in order to reduce the frequency of cannibalistic behavior at the fry stage. Video data analysis using bioimage informatics quantified the frequency of aggressive behavior, indicating a significant 46% reduction (P = 0.0229) in the frequency of cannibalistic behavior than in wild type. Furthermore, in the V1a2 KO strain, the frequency of collisions with the wall and oxygen consumption also decreased. Overall, the manageable and calm phenotype reported here can potentially contribute to the development of a stable and sustainable marine product.

Multicenter retrospective study of nivolumab for recurrent/metastatic oral squamous cell carcinoma.

OBJECTIVES: Immunotherapy with nivolumab for patients with recurrent/metastatic oral squamous cell carcinoma has not been evaluated. Here, we aimed to examine the efficacy, safety, and prognostic factors of nivolumab in these patients. MATERIALS AND METHODS: This multicenter retrospective observational study involved patients who received nivolumab between April 2017 and June 2019. The patient characteristics were evaluated for association with progression-free and overall survival. Progression-free and overall survival rates were calculated; parameters that were significant in the univariate analysis were used as explanatory variables. Independent factors for progression-free and overall survival were identified using multivariate analysis. RESULTS: Totally, 143 patients were included. The overall response and disease control rates were 27.3% and 46.2%, respectively. The median, 1- and 2-year progression-free survival rates were 2.7 months, 25.4%, and 19.2%, respectively; those for overall survival were 11.2 months, 47.3%, and 33.6%, respectively. The independent factors affecting progression-free survival were performance status and immune-related adverse event occurrence, whereas those affecting overall survival were performance status, target disease, and number of previous lines of systemic cancer therapy. Eight patients reported grade ≥3 immune-related adverse events. CONCLUSION: Nivolumab was effective for recurrent/metastatic oral squamous cell carcinoma treatment and was well tolerated by patients.

Simultaneous Enhancement of iron Deficiency Tolerance and Iron Accumulation in Rice by Combining the Knockdown of OsHRZ Ubiquitin Ligases with the Introduction of Engineered Ferric-chelate Reductase.

Iron is an essential micronutrient for living organisms, but its solubility is extremely low under alkaline conditions. Plants often suffer from iron deficiency chlorosis in calcareous soils, which consist of approximately 30% of the world's cultivated area, severely limiting plant productivity. Iron deficiency anemia is also a widespread problem in humans, especially in Asian and African people who take up iron mainly from staple foods containing low iron concentrations. Transgenic manipulation of genes involved in plant iron uptake, translocation, and storage has made improvements in enhancing iron deficiency tolerance or iron accumulation in edible parts, but these two properties have been characterized separately. We previously produced transgenic rice lines, with concomitant improvement of iron deficiency tolerance and grain iron accumulation by knocking-down OsHRZ ubiquitin ligases, which negatively regulate iron deficiency response and iron accumulation in rice. In the present report, we aimed to further improve the iron deficiency tolerance and grain iron accumulation of OsHRZ knockdown rice by the simultaneous introduction of the engineered ferric-chelate reductase gene Refre1/372 under the control of the OsIRT1 promoter for further enhancement of iron uptake. We obtained several transgenic rice lines with repressed OsHRZ expression and induced Refre1/372 expression. These lines showed a variable degree of iron deficiency tolerance in calcareous soils, with increased iron accumulation in brown seeds under both iron-deficient and iron-sufficient soil cultures. Selected OsHRZ knockdown plus Refre1/372 lines showed similar or better growth compared with that of singly introduced OsHRZ knockdown or Refre1/372 lines in calcareous soils under both non-submerged and submerged conditions. After submerged calcareous soil cultivation, these OsHRZ knockdown plus Refre1/372 lines accumulated 2.5-4.3 times and 17-23 times more iron concentrations than that of non-transformants in brown rice and straw, respectively, which was comparable or superior to a single OsHRZ knockdown line. Our results indicate that the combined introduction of OsHRZ knockdown and OsIRT1 promoter-Refre1/372 is highly effective in further improving the iron deficiency tolerance without compromising the iron accumulation of the OsHRZ knockdown effects.

Protocol for a multicentre, prospective observational study of elective neck dissection for clinically node-negative oral tongue squamous cell carcinoma (END-TC study).

INTRODUCTION: In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis is suspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCC remains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC. METHODS AND ANALYSIS: This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoints are 3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias. ETHICS AND DISSEMINATION: This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the corresponding author on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: UMIN000027875.

Inhaled volatile ß-caryophyllene is incorporated into the aortic wall and attenuates nicotine-induced aorta degeneration via a CB2 receptor-dependent pathway.

ß-caryophyllene (BCP) is a volatile bicyclic sesquiterpenoid found in essential oils obtained from several spices such as black pepper, oregano, basil, rosemary, cinnamon, and clove. BCP is a selective agonist of cannabinoid receptor 2 (CB2 receptor), and orally administered BCP exhibits various biological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, it is still unclear how volatile BCP affects living organisms. We previously reported that inhaled BCP is transferred to sera and organs in mice; additionally, metabolomic analysis revealed inhaled BCP affect the dynamics of metabolites in the livers of mice. These data suggest that inhaled BCP may affect several biological activities by stimulating biological systems. In this study, we evaluated the effects of BCP inhalation on nicotine-induced degeneration of the aortic wall. In the group of mice which inhaled volatile BCP, nicotine-induced increases in elastic fiber degradation and matrix metalloproteinase-2 (MMP-2)-positive areas were attenuated. In addition, BCP improved the nicotine-induced stiffness of aortae and vulnerability to aortic rupture. In cultured aortae, the suppressive effects of BCP were inhibited by the CB2 receptor inhibitor AM630. These results suggest that inhaled BCP is incorporated into the aortic wall and prevents nicotine-induced degeneration of the aorta via a CB2 receptor-dependent pathway.

The basic leucine zipper transcription factor OsbZIP83 and the glutaredoxins OsGRX6 and OsGRX9 facilitate rice iron utilization under the control of OsHRZ ubiquitin ligases.

Under low iron availability, plants induce the expression of various genes for iron uptake and translocation. The rice (Oryza sativa) ubiquitin ligases OsHRZ1 and OsHRZ2 cause overall repression of these iron-related genes at the transcript level, but their protein-level regulation is unclear. We conducted a proteome analysis to identify key regulators whose abundance was regulated by OsHRZs at the protein level. In response to iron deficiency or OsHRZ knockdown, many genes showed differential regulation between the transcript and protein levels, including the TGA-type basic leucine zipper transcription factor OsbZIP83. We also identified two glutaredoxins, OsGRX6 and OsGRX9, as OsHRZ-interacting proteins in yeast and plant cells. OsGRX6 also interacted with OsbZIP83. Our in vitro degradation assay suggested that OsbZIP83, OsGRX6 and OsGRX9 proteins are subjected to 26S proteasome- and OsHRZ-dependent degradation. Proteome analysis and our in vitro degradation assay also suggested that OsbZIP83 protein was preferentially degraded under iron-deficient conditions in rice roots. Transgenic rice lines overexpressing OsGRX9 and OsbZIP83 showed improved tolerance to iron deficiency. Expression of iron-related genes was affected in the OsGRX9 and OsGRX6 knockdown lines, suggesting disturbed iron utilization and signaling. OsbZIP83 overexpression lines showed enhanced expression of OsYSL2 and OsNAS3, which are involved in internal iron translocation, in addition to OsGRX9 and genes related to phytoalexin biosynthesis and the salicylic acid pathway. The results suggest that OsbZIP83, OsGRX6 and OsGRX9 facilitate iron utilization downstream of the OsHRZ pathway.

Palatoplasty for the Patient With Campomelic Dysplasia-Report of a Case and Review of the Literature.

Campomelic dysplasia (CMPD) is a skeletal disorder resulting from SOX9 gene mutations. Palatoplasty is rare due to a high lethality rate in infants from respiratory distress. Our patient had characteristic symptoms of CMPD, including short bowed limbs, macrocephaly, low-set ears, short palpebral fissures, hypertelorism, a flat nasal bridge, a long philtrum, micrognathia, and a cleft palate. We performed a Furlow palatoplasty when the patient was 2 years 9 months of age, after respiratory conditions had stabilized. We reviewed the literature of CMPD cases that underwent palatoplasty and discussed the optimal timing and surgical methods.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Estimation of tuna population by the improved analytical pipeline of unique molecular identifier-assisted HaCeD-Seq (haplotype count from eDNA).

Many studies have investigated the ability to identify species from environmental DNA (eDNA). However, even when individual species are identified, the accurate estimation of their abundances by traditional eDNA analyses has been still difficult. We previously developed a novel analytical method called HaCeD-Seq (Haplotype Count from eDNA), which focuses on the mitochondrial D-loop sequence. The D-loop is a rapidly evolving sequence and has been used to estimate the abundance of eel species in breeding water. In the current study, we have further improved this method by applying unique molecular identifier (UMI) tags, which eliminate the PCR and sequencing errors and extend the detection range by an order of magnitude. Based on this improved HaCeD-Seq pipeline, we computed the abundance of Pacific bluefin tuna (Thunnus orientalis) in aquarium tanks at the Tokyo Sea Life Park (Kasai, Tokyo, Japan). This tuna species is commercially important but is at high risk of resource depletion. With the developed UMI tag method, 90 out of 96 haplotypes (94%) were successfully detected from Pacific bluefin tuna eDNA. By contrast, only 29 out of 96 haplotypes (30%) were detected when UMI tags were not used. Our findings indicate the potential for conducting non-invasive fish stock surveys by sampling eDNA.

Development of a mugineic acid family phytosiderophore analog as an iron fertilizer.

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Roles of subcellular metal homeostasis in crop improvement.

Improvement of crop production in response to rapidly changing environmental conditions is a serious challenge facing plant breeders and biotechnologists. Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrients for plant growth and reproduction. These minerals are critical to several cellular processes including metabolism, photosynthesis, and cellular respiration. Regulating the uptake and distribution of these minerals could significantly improve plant growth and development, ultimately leading to increased crop production. Plant growth is limited by mineral deficiency, but on the other hand, excess Fe, Mn, Cu, and Zn can be toxic to plants; therefore, their uptake and distribution must be strictly regulated. Moreover, the distribution of these metals among subcellular organelles is extremely important for maintaining optimal cellular metabolism. Understanding the mechanisms controlling subcellular metal distribution and availability would enable development of crop plants that are better adapted to challenging and rapidly changing environmental conditions. Here, we describe advances in understanding of subcellular metal homeostasis, with a particular emphasis on cellular Fe homeostasis in Arabidopsis and rice, and discuss strategies for regulating cellular metabolism to improve plant production.

Iron deficiency-inducible peptide-coding genes OsIMA1 and OsIMA2 positively regulate a major pathway of iron uptake and translocation in rice.

Under low iron (Fe) availability, plants transcriptionally induce various genes responsible for Fe uptake and translocation to obtain adequate amounts of Fe. Although transcription factors and ubiquitin ligases involved in these Fe deficiency responses have been identified, the mechanisms coordinating these pathways have not been clarified in rice. Recently identified Fe-deficiency-inducible IRON MAN (IMA)/FE UPTAKE-INDUCING PEPTIDE (FEP) positively regulates many Fe-deficiency-inducible genes for Fe uptake in Arabidopsis. Here, we report that the expression of two IMA/FEP genes in rice, OsIMA1 and OsIMA2, is strongly induced under Fe deficiency, positively regulated by the transcription factors IDEF1, OsbHLH058, and OsbHLH059, as well as OsIMA1 and OsIMA2 themselves, and negatively regulated by HRZ ubiquitin ligases. Overexpression of OsIMA1 or OsIMA2 in rice conferred tolerance to Fe deficiency and accumulation of Fe in leaves and seeds. These OsIMA-overexpressing rice exhibited enhanced expression of all of the known Fe-deficiency-inducible genes involved in Fe uptake and translocation, except for OsYSL2, a Fe-nicotianamine transporter gene, in roots but not in leaves. Knockdown of OsIMA1 or OsIMA2 caused minor effects, including repression of some Fe uptake- and translocation-related genes in OsIMA1 knockdown roots. These results indicate that OsIMA1 and OsIMA2 play key roles in enhancing the major pathway of the Fe deficiency response in rice.

Defects in the rice aconitase-encoding OsACO1 gene alter iron homeostasis.

KEY MESSAGE: Rice aconitase gene OsACO1 is involved in the iron deficiency-signaling pathway for the expression of iron deficiency-inducible genes, either thorough enzyme activity or possible specific RNA binding for post-transcriptional regulation. Iron (Fe) is an essential element for virtually all living organisms. When plants are deficient in Fe, Fe acquisition systems are activated to maintain Fe homeostasis, and this regulation is mainly executed at the gene transcription level. Many molecules responsible for Fe uptake, translocation, and storage in plants have been identified and characterized. However, how plants sense Fe status within cells and then induce a transcriptional response is still unclear. In the present study, we found that knockdown of the OsACO1 gene, which encodes an aconitase in rice, leads to the down-regulation of selected Fe deficiency-inducible genes involved in Fe uptake and translocation in roots, and a decrease in Fe concentration in leaves, even when grown under Fe-sufficient conditions. OsACO1 knockdown plants showed a delayed transcriptional response to Fe deficiency compared to wild-type plants. In contrast, overexpression of OsACO1 resulted in the opposite effects. These results suggest that OsACO1 is situated upstream of the Fe deficiency-signaling pathway. Furthermore, we found that the OsACO1 protein potentially has RNA-binding activity. In vitro screening of RNA interactions with OsACO1 revealed that RNA potentially forms a unique stem-loop structure that interacts with OsACO1 via a conserved GGUGG motif within the loop structure. These results suggest that OsACO1 regulate Fe deficiency response either thorough enzyme activity catalyzing isomerization of citrate, or specific RNA binding for post-transcriptional regulation.

Author Correction: Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HaCeD-Seq: a Novel Method for Reliable and Easy Estimation About the Fish Population Using Haplotype Count from eDNA.

It is common to count the numbers of specified fish in the field after speciation of captured fish according to their morphology and to subject these counts to appropriate statistical analyses. In recent years, a non-invasive method to estimate the abundance of a particular fish species using environmental DNA (eDNA) has been developed. However, it is still difficult to determine accurate numbers of fish species using such method. We predict that the estimation of individuals of certain fish species in the field is more accurate and easier by using haplotypes of DNA in the fast evolutionary region. Therefore, we focused on the regulatory region (D-loop) in mitochondrial DNA, which is known to have a high genetic variation at the intraspecific level of the targeting eel. We investigated haplotype diversity in eel at first and then determined the number of D-loop haplotypes contained in their exfoliated cells in breeding water. Finally, we developed a novel analytical method, HaCeD-Seq, to estimate the number of individuals based on the abovementioned data.

Enhancement of Iron Acquisition in Rice by the Mugineic Acid Synthase Gene With Ferric Iron Reductase Gene and OsIRO2 Confers Tolerance in Submerged and Nonsubmerged Calcareous Soils.

Iron (Fe) is an essential micronutrient for plants. Plants encounter Fe deficiency when grown in calcareous soil with low Fe availability, leading to reduced crop yield and agricultural problem. Rice acquires Fe from the soil via Strategy I-related system (ferrous ion uptake by OsIRT1) and Strategy II system (ferric ion uptake by chelation). However, rice plants have a weak ability in Fe(III) reduction and phytosiderophore secretion. We previously produced an Fe deficiency-tolerant rice harboring OsIRT1 promoter-refre1/372 (for higher Fe(III) reductase ability) and a 35S promoter-OsIRO2 (for higher phytosiderophore secretion). In this study, we produced a new Fe deficiency-tolerant rice by the additional introduction of a barley IDS3 genome fragment with refre1/372 and OsIRO2 (named as IRI lines) for further enhancement in Strategy II phytosiderophore productivity and better growth performance in various environments. Our results show that an enhanced tolerance was observed in OsIRO2 introduced line at the early growth stage, refre1/372 introduced line in the late stage, and RI line in all stages among five types of cultivation method. Moreover, we demonstrated that new IRI rice lines exhibited enhanced tolerance to Fe deficiency compared to nontransgenic (NT) rice and rice lines harboring the overexpressing OsIRO2 or the IDS3 fragment under submerged calcareous soil. The yields of IRI lines were ninefold higher than the NT line. Furthermore, under Fe-limited nonsubmerged calcareous soil condition (a new cultivation condition), IRI lines also conferred enhanced tolerance than NT, lines introducing only the OsIRT1 promoter-refre1/372 or overexpressing OsIRO2, and lines harboring both. Our results demonstrate that further enhancement of the Strategy II Fe uptake system by the mugineic acid synthase gene in addition to Fe uptake by enhanced ferric Fe reduction and phytosiderophore production in rice contributes Fe deficiency tolerance and broaden its utility in calcareous soil cultivation under paddy or nonpaddy field conditions.