The effect of tea catechins on the forensic identification of urine: Urine camouflage to evade drug tests.

BACKGROUND: We encountered a urine sample suspected of being mixed with tea, submitted by a suspect attempting to camouflage illegal drugs. Although urine should turn reddish-pink during a urea test with p-Dimethylaminocinnamaldehyde (DAC), this suspect's sample exhibited a blue coloration when tested with DAC. AIM: Our aim was to examine the influence and mechanism of green tea on various urine identification tests. RESULTS: Our examination revealed that DAC forms a compound with the urea in urine, resulting in a reddish pink coloration with a molecular weight of 217. However, it has been reported that DAC binds to polyphenols such as catechin. In the case of catechin, DAC binds to the C8 position, forming a compound that exhibits the highest absorption at 640 nm and appears blue. we investigated the effect of urine from volunteers who had consumed a large amount of catechin on the urea test with DAC. Additionally, we carried out quantitative analysis of catechin in urine by LC-MS/MS after enzymatic treatment with ß-glucuronidase. The concentration of urinary excreted catechin reached its peak approximately 3 to 4 h after ingestion. During the DAC test, urine samples collected 3 to 4 h after catechin ingestion displayed a bluish pink color, but not the blue color observed in the original suspect sample. CONCLUSION: This study investigated the impact of catechin on urine tests, revealing that a blue color in the DAC test indicates a high likelihood of camouflage by the suspect.

A validated method for the separation of ethyl glucoside isomers by gas chromatography-tandem mass spectrometry and quantitation in human whole blood and urine.

Ethyl glucoside (EG) is present in Japanese sake in high concentrations, and can be found in other alcoholic beverages like beer and wine in varying amounts. EG exists as alpha (α) and beta (ß) isomers, and the concentrations and ratios of these isomers differ depending on the alcoholic beverage. Herein, we report a validated analysis method for the separation of EG isomers in human whole blood and urine, by GC-MS/MS. Whole blood and urine samples were deproteinized and interferences removed by weak cation exchange cartridges. The target analytes were acetylated using acetic anhydride and pyridine by microwave-accelerated derivatization. Separation was performed using tandem columns, with detection in the multiple reaction monitoring (MRM) mode. The MRM transitions for all compounds were m/z 157.0 > 115.1 for the quantifying transition, and m/z 157.0 > 73.1 and m/z 141.0 > 81.0 for the qualifying transitions. Assay validation included linearity, LOD and LLOQ, bias, within-run and between-run precision, stability, and dilution integrity. Baseline separation of the 2 isomers was achieved with linear calibration (r2 > 0.99) across the calibration range 0.625 to 50 µg/mL for both α- and ß-EG in both whole blood and urine. The validated method was then applied to actual human whole blood and urine samples collected at autopsy, as well as relevant alcoholic beverage samples. The quantitation of EG isomers could benefit the forensic toxicology community by acting as markers for recent alcoholic beverage consumption.

A large interhemispheric glioependymal cyst associated with partial defect of the corpus callosum in an elderly man.

Interhemispheric cysts are congenital, and usually present symptoms during childhood. However, they are occasionally detected in adults. These cystic lesions are sometimes associated with defects of the corpus callosum. Although defects of the corpus callosum by themselves do not present clinical symptoms, they are often accompanied by other brain malformations. A man in his late 60s was found dead at the scene of a fire. At autopsy, his brain weighed 1223 g and had a large interhemispheric cyst, measuring 5.5 × 4.5 × 4.0 cm in size. The cyst contained clear fluid but was not connected to the ventricular system. On slices of the cerebrum, the corpus callosum did not connect the right and left cerebral hemispheres, and the right lateral ventricle was dilated. By the existence of the cyst, compressed by the cyst, the hemispheres were displaced on either side. Histologically, the cerebral parenchyma around the cyst was slightly edematous but structurally normal. Immunohistochemically, both glial fibrillary acidic protein and podoplanin were expressed in the cystic components. Thus, the cystic lesion was diagnosed as a glioependymal cyst. In this case, because the cyst was located at the interhemispheric space between the right and left frontal lobes, the individual experienced no obvious symptoms, despite its large size. The individual's brain malformations included the partial defect of the corpus callosum and the cyst. The dilation of the right lateral ventricle was considered to result from the location of the cyst. Under the influence of the cyst, the third ventricle was displaced downward, and one or both of the interventricular foramen were obstructed. The decedent had burns over his whole body. Burns to the epiglottis and soot in the airway were also observed. Volatile hydrocarbons, such as benzene and styrene, were detected in the blood. The percentage of carboxyhemoglobin levels in a total of hemoglobin levels were 19-25%. Therefore, the individual's cause of death was established as death by fire.

Alternative Transoral Application of Nasopharyngeal Airways.

Transoral application of a nasopharyngeal airway (NPA) is a novel technique for difficult airway management. Clinically, it is an effective alternative for use in nonintubated dental cases under total intravenous anesthesia. This technique can help improve oxygenation and ventilation in clinical situations in which the conventional use of NPAs is ineffective, such as in patients who have findings of obesity; mandibular retrognathia or hypoplasia; maxillary hypoplasia; macroglossia; nasal obstruction secondary to hypertrophic tonsillar, adenoid, and/or lymphoid tissues or nasal polyps; known unusual nasal anatomy (eg, septal deviation); high risk of prolonged epistaxis (eg, patients on anticoagulants); or those who demonstrate mouth-breathing behaviors during deep sedation/nonintubated general anesthesia. After ensuring proper supraglottic placement, the transorally positioned NPA can be further secured with the use of tape for the duration of the dental procedure. Unlike an oropharyngeal airway, this simple and cost-efficient technique facilitates intraoral access for dental treatment.

New aspects of iron-copper crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana.

Iron (Fe) and copper (Cu) are essential micronutrients for energy metabolism and reactive oxygen species (ROS) scavenging. Some Cu-containing proteins can be substituted with Fe-containing proteins, and vice versa, while several Arabidopsis genes are regulated by both metals. Few details of how plants coordinate Fe-Cu crosstalk are known. Gene expression was measured in the roots and rosettes of Fe, Cu, and simultaneously Fe and Cu deficient WT plants and a mutant of the Cu-uptake transcription factor SPL7. The spl7 mutant accumulated excess Fe under normal conditions, and lower Fe supply rescued the growth phenotype and normalized the Fe : Cu ratios. Most Fe regulated genes were expressed similarly in the WT and spl7 mutant, although at higher fold-change levels in spl7 mutants. Expression patterns indicated that both SPL7 and the FIT Fe uptake transcription factor influenced the expression of many key Fe uptake genes. Most notably, the newly discovered IMA/FEP genes and the subgroup Ib bHLH genes, which are upstream of Fe uptake responses, were repressed in the WT under Cu deficiency. Several AP2/ethylene response factor (AP2/ERF) genes and other redox homeostasis network genes were derepressed in spl7 mutants. Together, we present new information about Fe-Cu crosstalk in plants that could be applied for developing abiotic stress tolerant crops.

Genome-Wide Association Study Reveals Novel Genomic Regions Associated with 10 Grain Minerals in Synthetic Hexaploid Wheat.

Synthetic hexaploid wheat (SHW; Triticum durum L. × Aegilops tauschii Coss.) is a means of introducing novel genes/genomic regions into bread wheat (T. aestivum L.) and a potential genetic resource for improving grain mineral concentrations. We quantified 10 grain minerals (Ca, Cd, Cu, Co, Fe, Li, Mg, Mn, Ni, and Zn) using an inductively coupled mass spectrometer in 123 SHWs for a genome-wide association study (GWAS). A GWAS with 35,648 single nucleotide polymorphism (SNP) markers identified 92 marker-trait associations (MTAs), of which 60 were novel and 40 were within genes, and the genes underlying 20 MTAs had annotations suggesting a potential role in grain mineral concentration. Twenty-four MTAs on the D-genome were novel and showed the potential of Ae. tauschii for improving grain mineral concentrations such as Ca, Co, Cu, Li, Mg, Mn, and Ni. Interestingly, the large number of novel MTAs (36) identified on the AB genome of these SHWs indicated that there is a lot of variation yet to be explored and to be used in the A and B genome along with the D-genome. Regression analysis identified a positive correlation between a cumulative number of favorable alleles at MTA loci in a genotype and grain mineral concentration. Additionally, we identified multi-traits and stable MTAs and recommended 13 top 10% SHWs with a higher concentration of beneficial grain minerals (Cu, Fe, Mg, Mn, Ni, and Zn), a large number of favorable alleles compared to low ranking genotypes and checks that could be utilized in the breeding program for the genetic biofortification. This study will further enhance our understanding of the genetic architecture of grain minerals in wheat and related cereals.

Association between cytochrome P450 2D6 polymorphisms and body fluid methamphetamine concentrations in Japanese forensic autopsy cases.

Methamphetamine (MA) is an illicit stimulant that affects the central nervous system. Cytochrome P450 2D6 (CYP2D6) plays an important role in MA metabolism. Numerous allelic variants confer substantial variation in CYP2D6 activity among individuals. In the present study, we examined the frequencies of CYP2D6 alleles, including CYP2D6*1, *2, *4, *5, *10, *14A, *14B, *18, and *36, and multiplication, in 82 forensic autopsy cases of MA abusers and 567 autopsy cases in which MA was not detected (controls). Ultrarapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) phenotypes were predicted from CYP2D6 genotypes. Of MA abusers, 64 subjects were predicted to be EM, 17 were IM, and 1 was UM. No MA abuser had the predicted PM phenotype. No significant differences in CYP2D6 phenotype frequencies were found between MA abusers and controls. MA and amphetamine (AMP) concentrations were measured in the right heart blood, left heart blood, peripheral external iliac blood, urine, pericardial fluid, and bone marrow of MA abusers. MA concentrations in urine and bone marrow were significantly higher in IM than in EM. AMP concentration was not associated with CYP2D6 phenotype in any body fluid. These results suggest that the MA concentration in body fluids is influenced by CYP2D6 phenotypes in the Japanese population.

Gene Expression Profiling of Iron Deficiency Chlorosis Sensitive and Tolerant Soybean Indicates Key Roles for Phenylpropanoids under Alkalinity Stress.

Alkaline soils comprise 30% of the earth and have low plant-available iron (Fe) concentration, and can cause iron deficiency chlorosis (IDC). IDC causes soybean yield losses of $260 million annually. However, it is not known whether molecular responses to IDC are equivalent to responses to low iron supply. IDC tolerant and sensitive soybean lines provide a contrast to identify specific factors associated with IDC. We used RNA-seq to compare gene expression under combinations of normal pH (5.7) or alkaline pH (7.7, imposed by 2.5 mM bicarbonate, or pH 8.2 imposed by 5 mM bicarbonate) and normal (25 µM) or low (1 µM) iron conditions from roots of these lines. Thus, we were able to treat pH and Fe supply as separate variables. We also noted differential gene expression between IDC sensitive and tolerant genotypes in each condition. Classical iron uptake genes, including ferric-chelate reductase (FCR) and ferrous transporters, were upregulated by both Fe deficiency and alkaline stress, however, their gene products did not function well at alkaline pH. In addition, genes in the phenylpropanoid synthesis pathway were upregulated in both alkaline and low Fe conditions. These genes lead to the production of fluorescent root exudate (FluRE) compounds, such as coumarins. Fluorescence of nutrient solution increased with alkaline treatment, and was higher in the IDC tolerant line. Some of these genes also localized to previously identified QTL regions associated with IDC. We hypothesize that FluRE become essential at alkaline pH where the classical iron uptake system does not function well. This work could result in new strategies to screen for IDC tolerance, and provide breeding targets to improve crop alkaline stress tolerance.

Giant intracranial arteriovenous malformation as the focus of epileptic seizures.

A man in his late thirties was found in a supine position in the hallway of his house. He had been diagnosed with epilepsy at approximately 20 years old. Since stopping treatment, epileptic events occurred more frequently and his condition deteriorated in the past 2 years. Autopsy revealed that head injuries were found on the left side of his head. A fracture from the left parietal bone to the anterior cranial fossa was also detected. A subdural hemorrhage (hematoma) spanned a wide range. A subarachnoid hemorrhage was also identified in the left parietal region. His brain weighed 1603 g, was edematous, and showed right uncal herniation. In the right cerebral hemisphere, a thick, enlarged blood vessel ran from the sagittal sinus. An egg-sized tumorous lesion of blood vessels was found on the bottom of the frontal lobe. This vascular lesion had formed between the sagittal sinus and right anterior cerebral artery. Pathologically, veins and arteries were found together, and, thus, this case was diagnosed as an arteriovenous malformation (AVM). No other pathological and toxicological findings were observed. Subdural hematoma, the cause of death, occurred from the fall to the floor. An epileptic seizure may have been the cause of the fall. AVM on his brain was considered to be the focal lesion of epileptic seizures.

Mapping and Characterization of the fefe Gene That Controls Iron Uptake in Melon (Cucumis melo L.).

Iron (Fe) deficiency in plants limits crop growth and productivity. Molecular mechanisms that plants use to sense and respond to Fe deficiency by coordinated expression of Fe-uptake genes are not fully understood. The C940-fe chlorotic melon (Cucumis melo) mutant known as fefe is unable to upregulate Fe-uptake genes, however, the FeFe gene had not been identified. In this study, we used two F2 mapping populations to map and identify the FeFe gene as bHLH38, a homolog of subgroup Ib bHLH genes from Arabidopsis thaliana that are involved in transcriptional regulation of Fe-uptake genes in partnership with the FIT gene. A Ty1-copia type retrotransposon insertion of 5.056 kb within bHLH38 is responsible for the defect in bHLH38 in fefe, based on sequencing and expression analysis. This retrotransposon insertion results in multiple non-functional transcripts expected to result in an altered and truncated protein sequence. Hairy root transformation of fefe plants using wild-type bHLH38 resulted in functional complementation of the chlorotic fefe phenotype. Using a yeast-2-hybrid assay, the transcription factor Fit interacted with the wild-type bHLH38 protein, but did not interact with the fefe bHLH38 protein, suggesting that heterodimer formation of Fit/bHLH38 to regulate Fe-uptake genes does not occur in fefe roots. The second subgroup Ib bHLH gene in the melon genome is not functionally redundant to bHLH38, in contrast to Arabidopsis where four subgroup Ib bHLH genes are functionally redundant. Whereas the Arabidopsis bHLH transcript levels are upregulated by Fe deficiency, melon bHLH38 was not regulated at the transcript level. Thus, the fefe mutant may provide a platform for studying bHLH38 genes and proteins from other plant species.

An unexpected death due to massive ascites and a giant mucinous ovarian cystadenoma.

A female in her thirties fell face down in her room. She was motionless when her sister found her. She was transported to the hospital by ambulance and was in a state of cardiopulmonary arrest on admission. She did not respond to resuscitation. Her abdomen had started to swell 3years before her death. An autopsy was performed to clarify the decedent's cause of death. She was 172cm tall and weighed 146kg. Her maximum abdominal girth was 172.1cm. A subcutaneous hemorrhage measuring 4.5cm in diameter was observed in the epigastric region. The abdominal cavity contained brownish ascites (54.1L). The left ovary was markedly swollen, and the combined weight of the uterus and right ovary was more than 13.0kg. A left ovarian tumor consisting of serous and mucinous cysts was detected. There were no metastatic lesions in the peritoneum or other organs. She might have suffered circulatory disturbance caused by the ascites and ovarian tumor. Moreover, being in a prone position would have resulted in an increase in intra-abdominal pressure, further exacerbating her circulatory problems. Therefore, her cause of death was considered to be circulatory failure caused by significant ascites and a large ovarian tumor.

Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis.

Iron (Fe) is an essential mineral that has low solubility in alkaline soils, where its deficiency results in chlorosis. Whether low Fe supply and alkaline pH stress are equivalent is unclear, as they have not been treated as separate variables in molecular physiological studies. Additionally, molecular responses to these stresses have not been studied in leaf and root tissues simultaneously. We tested how plants with the Strategy I Fe uptake system respond to Fe deficiency at mildly acidic and alkaline pH by measuring root ferric chelate reductase (FCR) activity and expression of selected Fe uptake genes and riboflavin synthesis genes. Alkaline pH increased cucumber (Cucumis sativus L.) root FCR activity at full Fe supply, but alkaline stress abolished FCR response to low Fe supply. Alkaline pH or low Fe supply resulted in increased expression of Fe uptake genes, but riboflavin synthesis genes responded to Fe deficiency but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but alkaline stress blocked up-regulation of these genes in Fe-deficient leaves. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These results suggest a model implicating shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots.

Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.).

Hard winter wheat (Triticum aestivum L.) is a major crop in the Great Plains of the United States, and our previous work demonstrated that wheat genotypes vary for grain cadmium accumulation with some exceeding the CODEX standard (0.2 mg kg(-1)). Previous reports of cadmium distribution in flour milling fractions have not included high cadmium grain. This study measured the distribution of cadmium, zinc, and iron in flour and bran streams from high cadmium (0.352 mg kg(-1)) grain on a pilot mill that produced 12 flour and four bran streams. Recovery in flour was substantially greater for cadmium (50%) than for zinc (31%) or iron (22%). Cadmium, zinc, and iron in the lowest mineral concentration flour stream, representing the purest endosperm fraction, were 52, 22, and 11%, respectively, of initial grain concentration. Our results indicate that, relative to zinc and iron, a greater proportion of cadmium is stored in the endosperm, the source of white flour.

Switchgrass (Panicum virgatum L) flag leaf transcriptomes reveal molecular signatures of leaf development, senescence, and mineral dynamics.

Switchgrass flag leaves can be expected to be a source of carbon to the plant, and its senescence is likely to impact the remobilization of nutrients from the shoots to the rhizomes. However, many genes have not been assigned a function in specific stages of leaf development. Here, we characterized gene expression in flag leaves over their development. By merging changes in leaf chlorophyll and the expression of genes for chlorophyll biosynthesis and degradation, a four-phase molecular roadmap for switchgrass flag leaf ontogeny was developed. Genes associated with early leaf development were up-regulated in phase 1. Phase 2 leaves had increased expression of genes for chlorophyll biosynthesis and those needed for full leaf function. Phase 3 coincided with the most active phase for leaf C and N assimilation. Phase 4 was associated with the onset of senescence, as observed by declining leaf chlorophyll content, a significant up-regulation in transcripts coding for enzymes involved with chlorophyll degradation, and in a large number of senescence-associated genes. Of considerable interest were switchgrass NAC transcription factors with significantly higher expression in senescing flag leaves. Two of these transcription factors were closely related to a wheat NAC gene that impacts mineral remobilization. The third switchgrass NAC factor was orthologous to an Arabidopsis gene with a known role in leaf senescence. Other genes coding for nitrogen and mineral utilization, including ureide, ammonium, nitrate, and molybdenum transporters, shared expression profiles that were significantly co-regulated with the expression profiles of the three NAC transcription factors. These data provide a good starting point to link shoot senescence to the onset of dormancy in field-grown switchgrass.

Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk.

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe-uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild-type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe-regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-deficient plants was independent of the normal Fe-uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe-regulated, and one was Cu-regulated. Simultaneous Fe and Cu deficiency synergistically up-regulated Fe-uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, and thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants.

Optimal copper supply is required for normal plant iron deficiency responses.

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. Understanding crosstalk between Fe and Cu nutrition could lead to strategies for improved growth on soils with low or excess metals, with implications for agriculture and phytoremediation. Here, we show that Cu and Fe nutrition interact to increase or decrease Fe and/or Cu accumulation in leaves and Fe uptake processes. Leaf Cu concentration increased under low Fe supply, while high Cu lowered leaf Fe concentration. Ferric reductase activity, an indicator of Fe demand, was inhibited at insufficient or high Cu supply. Surprisingly, plants grown without Fe were more susceptible to Cu toxicity.

Rosette iron deficiency transcript and microRNA profiling reveals links between copper and iron homeostasis in Arabidopsis thaliana.

Iron (Fe) is an essential plant micronutrient, and its deficiency limits plant growth and development on alkaline soils. Under Fe deficiency, plant responses include up-regulation of genes involved in Fe uptake from the soil. However, little is known about shoot responses to Fe deficiency. Using microarrays to probe gene expression in Kas-1 and Tsu-1 ecotypes of Arabidopsis thaliana, and comparison with existing Col-0 data, revealed conserved rosette gene expression responses to Fe deficiency. Fe-regulated genes included known metal homeostasis-related genes, and a number of genes of unknown function. Several genes responded to Fe deficiency in both roots and rosettes. Fe deficiency led to up-regulation of Cu,Zn superoxide dismutase (SOD) genes CSD1 and CSD2, and down-regulation of FeSOD genes FSD1 and FSD2. Eight microRNAs were found to respond to Fe deficiency. Three of these (miR397a, miR398a, and miR398b/c) are known to regulate transcripts of Cu-containing proteins, and were down-regulated by Fe deficiency, suggesting that they could be involved in plant adaptation to Fe limitation. Indeed, Fe deficiency led to accumulation of Cu in rosettes, prior to any detectable decrease in Fe concentration. ccs1 mutants that lack functional Cu,ZnSOD proteins were prone to greater oxidative stress under Fe deficiency, indicating that increased Cu concentration under Fe limitation has an important role in oxidative stress prevention. The present results show that Cu accumulation, microRNA regulation, and associated differential expression of Fe and CuSOD genes are coordinated responses to Fe limitation.

The effectiveness of injury-prevention programs in reducing the incidence of anterior cruciate ligament sprains in adolescent athletes.

CLINICAL SCENARIO: There is a high incidence of anterior cruciate ligament (ACL) injury in adolescents participating in pivoting sports such as soccer, basketball, and handball. Most ACL injuries in athletes are noncontact injuries, with a mechanism of sudden deceleration, change in direction, or landing from a jump. These mechanisms coupled with an increase in contraction of the quadriceps have been shown as risk factors for ACL injuries. Injuries to the ACL may require surgery, a long rehabilitation, and the potential for reinjury. Studies have shown reductions in lower extremity injury rates using training protocols that focus on landing mechanics, balance training, strength training, and/or agility training. There has been some thought that starting preventive training programs with adolescent athletes may be the most effective approach to reducing adolescent ACL injuries. FOCUSED CLINICAL QUESTION: Can lower extremity injury-prevention programs effectively reduce ACL injury rates in adolescent athletes?

Use of natural variation reveals core genes in the transcriptome of iron-deficient Arabidopsis thaliana roots.

Iron (Fe) is an essential mineral micronutrient for plants and animals. Plants respond to Fe deficiency by increasing root uptake capacity. Identification of gene networks for Fe uptake and homeostasis could result in improved crop growth and nutritional value. Previous studies have used microarrays to identify a large number of genes regulated by Fe deficiency in roots of three Arabidopsis ecotypes. However, a large proportion of these genes may be involved in secondary or genotype-influenced responses rather than in a universal role in Fe uptake or homeostasis. Here we show that a small percentage of the Fe deficiency transcriptome of two contrasting ecotypes, Kas-1 and Tsu-1, was shared with other ecotypes. Kas-1 and Tsu-1 had different timing and magnitude of ferric reductase activity upon Fe withdrawal, and different categories of overrepresented Fe-regulated genes. To gain insights into universal responses of Arabidopsis to Fe deficiency, the Kas-1 and Tsu-1 transcriptomes were compared with those of Col-0, Ler, and C24. In early Fe deficiency (24-48 h), no Fe-downregulated genes and only 10 upregulated genes were found in all ecotypes, and only 20 Fe-downregulated and 58 upregulated genes were found in at least three of the five ecotypes. Supernode gene networks were constructed to visualize conserved Fe homeostasis responses. Contrasting gene expression highlighted different responses to Fe deficiency between ecotypes. This study demonstrates the use of natural variation to identify central Fe-deficiency-regulated genes in plants, and identified genes with potential new roles in signalling during Fe deficiency.

Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain.

The NAM-B1 gene is a NAC transcription factor that affects grain nutrient concentrations in wheat (Triticum aestivum). An RNAi line with reduced expression of NAM genes has lower grain protein, iron (Fe), and zinc (Zn) concentrations. To determine whether decreased remobilization, lower plant uptake, or decreased partitioning to grain are responsible for this phenotype, mineral dynamics were quantified in wheat tissues throughout grain development. Control and RNAi wheat were grown in potting mix and hydroponics. Mineral (Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn) and nitrogen (N) contents of organs were determined at regular intervals to quantify the net remobilization from vegetative tissues and the accumulation of nutrients in grain. Total nutrient accumulation was similar between lines, but grain Fe, Zn, and N were at lower concentrations in the NAM knockdown line. In potting mix, net remobilization of N, Fe, and Zn from vegetative tissues was impaired in the RNAi line. In hydroponics with ample nutrients, net remobilization was not observed, but grain Fe and Zn contents and concentrations remained lower in the RNAi line. When Fe or Zn was withheld post-anthesis, both lines demonstrated remobilization. These results suggest that a major effect of the NAM genes is an increased efflux of nutrients from the vegetative tissues and a higher partitioning of nutrients to grain.