Search

Although drug-coated balloon (DCB) -based stent-less percutaneous coronary intervention (PCI) for de novo lesions has attracted more attention, outcomes of the DCB procedure for hemodialysis (HD) patients are reported to be inferior to those for non-HD patients, similarly to drug-eluting stent (DES). Recent several reports have shown that rotational atherectomy (RA) followed by DCB treatment (RA/DCB) could be an option of revascularization strategy particularly for calcified de novo lesions even in the new-generation DES era; however, efficacy of the RA/DCB procedure for HD patients remains unclear.A total of 47 consecutive cases (53 lesions) undergoing RA/DCB for de novo lesions were enrolled. According to the presence/absence of HD at baseline, the 47 cases were divided into the HD cases (n=16) and the non-HD cases (n=31), and the 53 lesions were divided into the HD lesions (n=20) and the non-HD lesions (n=33).The HD cases had a significantly lower prevalence of dyslipidemia and smoking than the non-HD cases. Final RA burr size, DCB diameter used, and angiographic success rate of PCI did not significantly differ between the 2 groups. Pre-procedural, post-procedural, and follow-up QCA parameters were also similar between the 2 groups. Twelve-month clinical outcomes were comparable between the 2 groups.Mid-term outcomes of stent-less PCI using RA/DCB for de novo lesions in HD patients might be comparable to those in non-HD patients, suggesting efficacy of pre-treatment of RA prior to DCB treatment in HD patients.

p-Phenoxyphenyl boronic acid (PPBo) is a specific inhibitor of auxin biosynthesis in Arabidopsis. We examined the inhibitory activity of PPBo in rice. The activity of OsYUCCA, a key enzyme for auxin biosynthesis, was inhibited by PPBo in vitro. The endogenous indole-3-acetic acid (IAA) level and the expression levels of auxin-response genes were significantly reduced in PPBo-treated rice seedlings, which showed typical auxin-deficiency phenotypes. Seminal root growth was promoted by 1 µM PPBo, which was reversed by co-treatment of IAA and PPBo. By contrast, the inhibition of root growth by 10 µM PPBo was not recovered by IAA. The root meristem morphology and cell division were restored by IAA at 60 µM, but that concentration may be too high to support root growth. In conclusion, PPBo is an inhibitor of auxin biosynthesis that targets YUCCA in rice.

Melting-flesh peaches produce large amounts of ethylene, resulting in rapid fruit softening at the late-ripening stage. In contrast, stony hard peaches do not soften and produce little ethylene. The indole-3-acetic acid (IAA) level in stony hard peaches is low at the late-ripening stage, resulting in low ethylene production and inhibition of fruit softening. To elucidate the mechanism of low IAA concentration in stony hard peaches, endogenous levels of IAA and IAA intermediates or metabolites were analysed by ultra-performance liquid chromatography-tandem mass spectrometry. Although the IAA level was low, the indole-3-pyruvic acid (IPyA) level was high in stony hard peaches at the ripening stage. These results indicate that YUCCA activity is reduced in ripening stony hard peaches. The expression of one of the YUCCA isogenes in peach, PpYUC11, was suppressed in ripening stony hard peaches. Furthermore, an insertion of a transposon-like sequence was found upstream of the PpYUC11 gene in the 5'-flanking region. Analyses of the segregation ratio of the stony hard phenotype and genotype in F1 progenies indicated that the transposon-inserted allele of PpYUC11, hd-t, correlated with the stony hard phenotype. On the basis of the above findings, we propose that the IPyA pathway (YUCCA pathway) is the main auxin biosynthetic pathway in ripening peaches of 'Akatsuki' and 'Manami' cultivars. Because IAA is not supplied from storage forms, IAAde novo synthesis via the IPyA pathway (YUCCA pathway) in mesocarp tissues is responsible for auxin generation to support fruit softening, and its disruption can lead to the stony hard phenotype.

Increasing grain yield is an endless challenge for cereal crop breeding. In barley (Hordeum vulgare), grain number is controlled mainly by Six-rowed spike 1 (Vrs1), which encodes a homeodomain leucine zipper class I transcription factor. However, little is known about the genetic basis of grain size. Here, we show that extreme suppression of lateral florets contributes to enlarged grains in deficiens barley. Through a combination of fine-mapping and resequencing of deficiens mutants, we have identified that a single amino acid substitution at a putative phosphorylation site in VRS1 is responsible for the deficiens phenotype. deficiens mutant alleles confer an increase in grain size, a reduction in plant height, and a significant increase in thousand grain weight in contemporary cultivated germplasm. Haplotype analysis revealed that barley carrying the deficiens allele (Vrs1.t1) originated from two-rowed types carrying the Vrs1.b2 allele, predominantly found in germplasm from northern Africa. In situ hybridization of histone H4, a marker for cell cycle or proliferation, showed weaker expression in the lateral spikelets compared with central spikelets in deficiens. Transcriptome analysis revealed that a number of histone superfamily genes were up-regulated in the deficiens mutant, suggesting that enhanced cell proliferation in the central spikelet may contribute to larger grains. Our data suggest that grain yield can be improved by suppressing the development of specific organs that are not positively involved in sink/source relationships.

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

Long intergenic noncoding RNAs (lincRNAs) play critical roles in transcriptional and post-transcriptional regulation of gene expression in a wide variety of organisms. Thousands of lincRNAs have been identified in plant genomes, although their functions remain mostly uncharacterized. Here, we report a genome-wide survey of lincRNAs involved in the response to low-nutrient conditions in Arabidopsis thaliana. We used RNA sequencing data derived from A. thaliana roots exposed to low levels of 12 different nutrients. Using bioinformatics approaches, 60 differentially expressed lincRNAs were identified that were significantly upregulated or downregulated under deficiency of at least one nutrient. To clarify their roles in nutrient response, correlations of expression patterns between lincRNAs and reference genes were examined across the 13 conditions (12 low-nutrient conditions and control). This analysis allowed us to identify lincRNA-RNA pairs with highly positive or negative correlations. In addition, calculating interaction energies of those pairs showed lincRNAs that may act as regulatory interactors; e.g. small interfering RNAs (siRNAs). Among them, trans-acting siRNA3 (TAS3), which is known to promote lateral root development by producing siRNA against Auxin response factor 2, 3, and 4, was revealed as a nitrogen (N)-responsive lincRNA. Furthermore, nitrate transporter 2 was identified as a potential target of TAS3-derived siRNA, suggesting that TAS3 participates in multiple pathways by regulating N transport and root development under low-N conditions. This study provides the first resource for candidate lincRNAs involved in multiple nutrient responses in plants.

Summary Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin-deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole-3-pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole-3-acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin-containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4-biphenylboronic acid (BBo) and 4-phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild-type Arabidopsis seedlings. Co-treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (Ki) of BBo and PPBo were 67 and 56 nm, respectively. In addition, PPBo did not interfere with the auxin response of auxin-marker genes when it was co-treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.

We previously reported that exogenous application of auxin to Arabidopsis seedlings resulted in downregulation of indole-3-acetic acid (IAA) biosynthesis genes in a feedback manner. In this study, we investigated the involvement of the SCF

We previously reported that exogenous application of auxin to Arabidopsis seedlings resulted in downregulation of indole-3-acetic acid (IAA) biosynthesis genes in a feedback manner. In this study, we investigated the involvement of the SCFTIR1/AFB-mediated signaling pathway in feedback regulation of the indole-3-pyruvic acid-mediated auxin biosynthesis pathway in Arabidopsis. Application of PEO-IAA, an inhibitor of the IAA signal transduction pathway, to wild-type seedlings resulted in increased endogenous IAA levels in roots. Endogenous IAA levels in the auxin-signaling mutants axr2-1, axr3-3, and tir1-1afb1-1afb2-1afb3-1 also increased. Furthermore, YUCCA (YUC) gene expression was repressed in response to auxin treatment, and expression of YUC7 and YUC8 increased in response to PEO-IAA treatment. YUC genes were also induced in auxin-signaling mutants but repressed in TIR1-overexpression lines. These observations suggest that the endogenous IAA levels are regulated by auxin biosynthesis in a feedback manner, and the Aux/IAA and SCFTIR1/AFB-mediated auxin-signaling pathway regulates the expression of YUC genes.

An improved understanding of the Ni root-to-shoot translocation mechanism in hyperaccumulators is necessary to increase Ni uptake efficiency for phytoextraction technologies. It has been presumed that an important aspect of Ni translocation and storage involves chelation with organic ligands. It has been reported that exposing several Ni hyperaccumulator species of Alyssum to Ni elicited a large increase in the histidine level of the xylem sap. In later studies it was shown that as time progressed the histidine:Ni ratio dropped considerably. Moreover, previous studies analyzed the relationship between Ni and ligands in plants that were exposed to Ni only for a few hours and therefore obtained results that are unlikely to represent field soils where plants are at steady-state Ni uptake. The aim of this study was to understand the quantitative relationship between Ni and organic ligands in the xylem sap of various Alyssum genotypes or species that reached steady-state Ni uptake after being exposed to Ni in either nutrient solution or serpentine soil for up to 6 weeks. Total Ni concentration, 17 amino acids, 9 organic acids, and nicotianamine were measured in xylem sap of 100-day old plants of Alyssum. Results showed that the concentration of Ni in xylem sap of various Alyssum genotypes was 10–100 fold higher than the concentration of histidine, malate, citrate, and nicotianamine, which were the predominant Ni ligands measured in the sap. When the physiology of the whole plant is taken into account, our results indicate that the concentration of organic chelators is too low to account for the complexation of all the Ni present in the xylem sap of Alyssum at steady-state Ni hyperaccumulation, and suggest that most of the Ni in xylem sap of this species is present as the hydrated cation.

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe-deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1-like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)-DMA. These results imply that in alkaline soil, Fe(III)-DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.

Summary The alteration of transcript structure contributes to transcriptome plasticity. In this study, we analyzed the genome-wide response of exon combination patterns to deficiencies in 12 different nutrients in Arabidopsis thaliana roots. RNA sequencing analysis and bioinformatics using a simulation survey revealed more than 600 genes showing varying exon combinations. The overlap between genes showing differential expression (DE) and genes showing differential exon combination (DC) was notably low. Additionally, gene ontology analysis showed that gene functions were not shared between the DE and DC genes, suggesting that the genes showing DC had different roles than those showing DE. Most of the DC genes were nutrient specific. For example, two homologs of the MYB transcription factor genes MYB48 and MYB59 showed differential alternative splicing only in response to low levels of potassium. Alternative splicing of those MYB genes modulated DNA-binding motifs, and MYB59 is reportedly involved in the inhibition of root elongation. Therefore, the increased abundance of MYB isoforms with an intact DNA-binding motif under low potassium may be involved in the active inhibition of root elongation. Overall, we provide global and comprehensive data for DC genes affected by nutritional deficiencies, which contribute to elucidating an unknown mechanism involved in adaptation to nutrient deficiency.

Peanut/maize intercropping is a sustainable and effective agroecosystem to alleviate iron-deficiency chlorosis. Using suppression subtractive hybridization from the roots of intercropped and monocropped peanut which show different iron nutrition levels, a peanut gene, AhNRAMP1, which belongs to divalent metal transporters of the natural resistance-associated macrophage protein (NRAMP) gene family was isolated. Yeast complementation assays suggested that AhNRAMP1 encodes a functional iron transporter. Moreover, the mRNA level of AhNRAMP1 was obviously induced by iron deficiency in both roots and leaves. Transient expression, laser microdissection, and in situ hybridization analyses revealed that AhNRAMP1 was mainly localized on the plasma membrane of the epidermis of peanut roots. Induced expression of AhNRAMP1 in tobacco conferred enhanced tolerance to iron deprivation. These results suggest that the AhNRAMP1 is possibly involved in iron acquisition in peanut plants.

Iron (Fe) deficiency is one of the major yield-limiting factors in peanut (Arachis hypogaea L.), especially when grown in calcareous soils. The soils of the Pothwar region in Pakistan are calcareous in nature, exposing peanut to Fe deficiency. The molecular mechanisms governing Fe deficiency response in peanut have not been fully revealed. We compared 4 locally important varieties of peanut to evaluate Fe deficiency responses at the molecular level. The expression profiles of 7 important genes including AhIRT1, AhFRO1, AhNRAMP1, AhYSL1, AhYSL3.1, AhYSL4, and AhYSL6 in roots and young and old leaves of local peanut varieties were investigated 19 days after Fe deficiency treatment. Significant differences were observed for gene expression patterns among these varieties. The expression of AhIRT1 was upregulated only in Banki and BARD-699. However, some of the genes, like AhFRO1, AhNRAMP1, and other genes of the YSL family, were upregulated in BARI 2000 roots and young and old leaves, which has been reported for uptake and transport of Fe. BARI 2000 and Chakori had more accumulation of Fe in young leaves and roots in Fe deficiency conditions as compared to other varieties. This suggests that other mechanisms of Fe uptake and transport may be more important than AhIRT1 to mitigate Fe deficiency in BARI 2000 and Chakori.

Iron (Fe) is an essential micronutrient for plants; however, despite its abundance in soils it is not readily available in neutral to alkaline soils. Plants secrete phenolics, such as protocatechuic acid (PCA) and caffeic acid (CA), to absorb and utilize precipitated apoplasmic Fe from root surfaces. However, the synthesis and secretion of phenolics have not been well characterized in plants. We have identified and characterized a rice (Oryza sativa L.) mutant with reduced amounts of PCA and CA in xylem sap and root exudates; hence we named it phenolics efflux zero 2 (pez2). PEZ2 localized to the plasma membrane in onion (Allium cepa L.) epidermal cells and transported PCA when expressed in Xenopus laevis oocytes. PEZ2 expression was observed in whole root near root tips. Similarly, strong expression was observed in leaves. In line with reduced amounts of PCA and CA in xylem sap, the xylem Fe concentration was also low in pez2 plants. These results suggest that PEZ2 is involved in solubilization of apopla...

Summary We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2′-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.

Oryza officinalis is an accessible alien donor for genetic improvement of rice. Comparison across a representative panel of Oryza species showed that the wild O. officinalis and cultivated O. sativa ssp. japonica have similar cold tolerance potentials. The possibility that either distinct or similar genetic mechanisms are involved in the low temperature responses of each species was addressed by comparing their transcriptional networks. General similarities were supported by shared transcriptomic signatures indicative of equivalent metabolic, hormonal, and defense status. However, O. officinalis has maintained an elaborate cold-responsive brassinosteroid-regulated BES1-network that appeared to have been fragmented in O. sativa. BES1-network is potentially important for integrating growth-related responses with physiological adjustments and defenses through the protection of photosynthetic machinery and maintenance of stomatal aperture, oxidative defenses, and osmotic adjustment. Equivalent physiological processes are functional in O. sativa but their genetic mechanisms are under the direct control of ABA-dependent, DREB-dependent and/or oxidative-mediated networks uncoupled to BES1. While O. officinalis and O. sativa represent long periods of speciation and domestication, their comparable cold tolerance potentials involve equivalent physiological processes but distinct genetic networks. BES1-network represents a novel attribute of O. officinalis with potential applications in diversifying or complementing other mechanisms in the cultivated germplasm.

Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs.

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 mul of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-beta-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.

Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.

The IPyA pathway, the major auxin biosynthesis pathway, is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels. The phytohormone auxin plays an important role in plant growth and development, and levels of active free auxin are determined by biosynthesis, conjugation, and polar transport. Unlike conjugation and polar transport, little is known regarding the regulatory mechanism of auxin biosynthesis. We discovered that expression of genes encoding indole-3-pyruvic acid (IPyA) pathway enzymes is regulated by elevated or reduced active auxin levels. Expression levels of TAR2, YUC1, YUC2, YUC4, and YUC6 were downregulated in response to synthetic auxins [1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)] exogenously applied to Arabidopsis thaliana L. seedlings. Concomitantly, reduced levels of endogenous indole-3-acetic acid (IAA) were observed. Alternatively, expression of these YUCCA genes was upregulated by the auxin biosynthetic inhibitor kynurenine in Arabidopsis seedlings, accompanied by reduced IAA levels. These results indicate that expression of YUCCA genes is regulated by active auxin levels. Similar results were also observed in auxin-overproduction and auxin-deficient mutants. Exogenous application of IPyA to Arabidopsis seedlings preincubated with kynurenine increased endogenous IAA levels, while preincubation with 2,4-D reduced endogenous IAA levels compared to seedlings exposed only to IPyA. These results suggest that in vivo conversion of IPyA to IAA was enhanced under reduced auxin levels, while IPyA to IAA conversion was depressed in the presence of excess auxin. Based on these results, we propose that the IPyA pathway is transcriptionally regulated through a negative feedback mechanism in response to active auxin levels.

In transcriptome experiments, the experimental conditions (e.g. mutants and/or treatments) cause transcriptional changes. Identifying experimental conditions that induce similar or opposite transcriptional changes can be useful to identify experimental conditions that affect the same biological process. AtCAST (http://atpbsmd.yokohama-cu.ac.jp) is a web-based tool to analyze the relationship between experimental conditions among transcriptome data. Users can analyze ‘user’s transcriptome data’ of a new mutant or a new chemical compound whose function remains unknown to generate novel biological hypotheses. This tool also allows for mining of related ‘experimental conditions’ from the public microarray data, which are pre-included in AtCAST. This tool extracts a set of genes (i.e. module) that show significant transcriptional changes and generates a network graph to present related transcriptome data. The updated AtCAST now contains data on >7,000 microarrays, including experiments on various stresses, mutants and chemical treatments. Gene ontology term enrichment (GOE) analysis is introduced to assist the characterization of transcriptome data. The new AtCAST supports input from multiple platforms, including the ‘Arabisopsis gene 1.1 ST array’, a new microarray chip from Affymetrix and RNA sequencing (RNA-seq) data obtained using next-generation sequencing (NGS). As a pilot study, we conducted microarray analysis of Arabidopsis under auxin treatment using the new Affymetrix chip, and then analyzed the data in AtCAST. We also analyzed RNA-seq data of the pifq mutant using AtCAST. These new features will facilitate analysis of associations between transcriptome data obtained using different platforms.

Iron (Fe) is an essential nutrient in both plants and humans. Fe deficiency on calcareous soil with low Fe availability is a major agricultural problem. Nicotianamine (NA) is one of the Fe chelator in plants, which is involved in metal translocation into seeds, and serves as an antihypertensive substance in humans. In this study, soybean plants overexpressing the barley NA synthase 1 (HvNAS1) gene driven by the constitutive CaMV 35S promoter were produced using Agrobacterium-mediated transformation. The transgenic soybean showed no growth defect and grew normally. The NA content of transgenic soybean seeds was up to four-fold greater than that of non-transgenic (NT) soybean seeds. The level of HvNAS1 expression was positively correlated with the amount of NA, and a high concentration of NA was maintained in the seeds in succeeding generations. The Fe concentration was approximately two-fold greater in transgenic soybean seeds than in NT soybean seeds. Furthermore, the transgenic soybeans showed tolerance to low Fe availability in calcareous soil. Our results suggested that increasing the NA content in soybean seeds by the overexpression of HvNAS1 offers potential benefits for both human health and agricultural productivity.

Brassinosteroids are plant steroid hormones that regulate plant organs and chloroplast development. The detailed molecular mechanism for plant development by BR signaling is yet to be revealed, and many points regarding the relationship between BR signaling and chloroplast development remain unknown. We identify here the dominant mutant Brz-insensitive-pale green3-1D (bpg3-1D) from the Arabidopsis FOX lines that show reduced sensitivity to the chlorophyll accumulation promoted by the BR biosynthesis inhibitor, Brassinazole (Brz), in the light. BPG3 encodes a novel chloroplast protein that is evolutionally conserved in bacteria, algae, and higher plants. The expression of BPG3 was induced by light and Brz. The inhibition of electron transport in photosystem II of the chloroplasts was detected in bpg3-1D. These results suggest that BPG3 played an important role in regulating photosynthesis in the chloroplast under BR signaling.

Several metals are essential nutrients for plants. However, they become toxic at high levels and deleteriously affect crop yield and quality. We recently reported the spatial gene expression profiles of iron (Fe)-deficient and cadmium (Cd)-stressed rice using laser microdissection and microarray analysis. The roots of Fe-deficient and Cd-stressed rice were separated into the vascular bundle (VB), cortex (Cor), and epidermis plus exodermis (EP). In addition, vascular bundles from new and old leaves at the lowest node, which are important for metal distribution, were analyzed separately (newDC and oldDC, respectively). Genes expressed in a tissue-specific manner in the VB, Cor, EP, newDC, and oldDC formed large clusters. The genes upregulated in all of the VB, Cor, and EP by Fe deficiency formed a substantial cluster that was smaller than the tissue-specific clusters. Significant numbers of genes expressed in newDC or oldDC were also expressed in VB in roots, suggesting that vascular bundles in the lowest nodes and roots have a partially common function. The expression patterns of transporter families involved in metal homeostasis were investigated, and members of each family were either expressed differentially in each tissue or showed different responses to Fe deficiency. One potassium transporter gene, OsHAK22, was upregulated by Fe deficiency in VB, Cor, and EP, suggesting that OsHAK22 is involved in potassium transport associated with mugineic acids secretion.

Iron (Fe) limitation is a widespread agricultural problem in calcareous soils and severely limits crop production. Iron Regulated Transporter 1 (IRT1) is a key component for Fe uptake from the soil in dicot plants. In this study, the peanut (Arachis hypogaea L.) AhIRT1 was introduced into tobacco and rice plants using an Fe-deficiency-inducible artificial promoter. Induced expression of AhIRT1 in tobacco plants resulted in accumulation of Fe in young leaves under Fe deficient conditions. Even under Fe-excess conditions, the Fe concentration was also markedly enhanced, suggesting that the Fe status did not affect the uptake and translocation of Fe by AhIRT1 in the transgenic plants. Most importantly, the transgenic tobacco plants showed improved tolerance to Fe limitation in culture in two types of calcareous soils. Additionally, the induced expression of AhIRT1 in rice plants also resulted in high tolerance to low Fe availability in calcareous soils.

Background Cis-acting elements are essential genomic sequences that control gene expression. In higher eukaryotes, a series of cis-elements function cooperatively. However, further studies are required to examine the co-regulation of multiple cis-elements on a promoter. The aim of this study was to propose a model of cis-element networks that cooperatively regulate gene expression in rice under iron (Fe) deficiency. Results We developed a novel clustering-free method, microarray-associated motif analyzer (MAMA), to predict novel cis-acting elements based on weighted sequence similarities and gene expression profiles in microarray analyses. Simulation of gene expression was performed using a support vector machine and based on the presence of predicted motifs and motif pairs. The accuracy of simulated gene expression was used to evaluate the quality of prediction and to optimize the parameters used in this method. Based on sequences of Oryza sativa genes upregulated by Fe deficiency, MAMA returned experimentally identified cis-elements responsible for Fe deficiency in O. sativa. When this method was applied to O. sativa subjected to zinc deficiency and Arabidopsis thaliana subjected to salt stress, several novel candidate cis-acting elements that overlap with known cis-acting elements, such as ZDRE, ABRE, and DRE, were identified. After optimization, MAMA accurately simulated more than 87% of gene expression. Predicted motifs strongly co-localized in the upstream regions of regulated genes and sequences around transcription start sites. Furthermore, in many cases, the separation (in bp) between co-localized motifs was conserved, suggesting that predicted motifs and the separation between them were important in the co-regulation of gene expression. Conclusions Our results are suggestive of a typical sequence model for Fe deficiency-responsive promoters and some strong candidate cis-elements that function cooperatively with known cis-elements.

Although the genes involved in metal homeostasis have been investigated over the past few decades, many genes related to metal homeostasis remain uncharacterized, and a comprehensive analysis of the expression of these genes is required. In the present study, we investigated the spatial gene expression profile of iron (Fe)-deficient and cadmium (Cd)-stressed Oryza sativa (rice) using laser microdissection and microarray analysis. Roots of Fe-deficient and Cd-stressed rice were separated into the vascular bundle, cortex, and epidermis plus exodermis. In addition, vascular bundles from new and old leaves at the lowest node, which are important for metal distribution, were analyzed separately. The spatial expression patterns were distinct in each tissue type. Fe deficiency and Cd stress also had significant effects on the transcriptomes, although these were less pronounced than the spatial effects. Genes encoding transporters involved in metal homeostasis, proteins associated with heavy metal detoxification, and phytohormone-related proteins were comprehensively investigated. Additionally, cis motifs involved in the regulation of these diverse expression changes in various tissue types were predicted. The spatial transcriptomes presented here provide novel insight into the molecular mechanisms of metal homeostasis.

To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T(2) polished seeds was sixfold higher and that in paddy field-grown T(3) polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes.

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe-deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1-like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)-DMA. These results imply that in alkaline soil, Fe(III)-DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.

Graminaceous plants acquire iron by secreting mugineic acid family phytosiderophores into the rhizosphere and taking up complexes of iron and phytosiderophores through YSL (yellow stripe 1-like) transporters. Rice OsYSL15 is a transporter of the iron(III)-2′-deoxymugineic acid complex. OsYSL16 has 85 % similarity to both OsYSL15 and the iron(II)-nicotianamine transporter OsYSL2. In the present study, we show that OsYSL16 functionally complemented a yeast mutant defective in iron uptake when grown on medium containing iron(III)-deoxymugineic acid, but not when grown on medium containing iron(II)-nicotianamine. OsYSL16-knockdown seedlings were smaller than wild-type seedlings when only iron(III)chloride was supplied as an iron source. The iron concentration in shoots of OsYSL16-knockdown plants was similar to that of the wild type; however, they showed more severe chlorosis than wild-type plants under iron-deficient conditions. Furthermore, OsYSL16-knockdown plants accumulated more iron in the vascular bundles of the leaves. Expression of the OsYSL16 promoter fused to the β-glucuronidase gene showed that OsYSL16 is expressed in the root epidermis and vascular bundles of whole plants. The expression was typically observed around the xylem. In the vascular bundles of unelongated nodes, it was detected in the xylem of old leaves and the phloem of new leaves. Graminaceous plants translocate iron from the roots to old leaves mainly via the xylem and to new leaves mainly via the phloem. Our results suggest that OsYSL16 plays a role in the allocation of iron(III)-deoxymugineic acid via the vascular bundles. Electronic supplementary material The online version of this article (doi:10.1007/s11103-012-9930-1) contains supplementary material, which is available to authorized users.

Iron deficiency is one of the major agricultural problems, as 30% of the arable land of the world is too alkaline for optimal crop production, rendering plants short of available iron despite its abundance. To take up apoplasmic precipitated iron, plants secrete phenolics such as protocatechuic acid (PCA) and caffeic acid. The molecular pathways and genes of iron uptake strategies are already characterized, whereas the molecular mechanisms of phenolics synthesis and secretion have not been clarified, and no phenolics efflux transporters have been identified in plants yet. Here we describe the identification of a phenolics efflux transporter in rice. We identified a cadmium-accumulating rice mutant in which the amount of PCA and caffeic acid in the xylem sap was dramatically reduced and hence named it phenolics efflux zero 1 (pez1). PEZ1 localized to the plasma membrane and transported PCA when expressed in Xenopus laevis oocytes. PEZ1 localized mainly in the stele of roots. In the roots of pez1, precipitated apoplasmic iron increased. The growth of PEZ1 overexpression lines was severely restricted, and these lines accumulated more iron as a result of the high solubilization of precipitated apoplasmic iron in the stele. We show that PEZ1 is responsible for an increase of PCA concentration in the xylem sap and is essential for the utilization of apoplasmic precipitated iron in the stele.

We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2'-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase ( NAS ) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3 , was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants ( OsNAS3-D1 ) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression.