Characterization of a putative mutant for iron homeostasis

Abstract only availableLittle is known about the genetics of iron homeostasis in plants. A novel genetic screen was used to identify mutants with alterations in iron homeostasis. Because Ferritin (Fer1) mRNA expression is upregulated by intracellular iron concentration in leaves, this gene can be used to predict intercellular iron concentrations in leaves. To identify mutants that over- or under-accumulate leaf iron, Arabidopsis was transformed with the reporter gene Green Fluorescent Protein (GFP) driven by the Fer1 promoter. Seed from this transgenic plant were mutagenized with EMS. The resulting M2 seed were screened for high or low GFP fluorescence relative to transgenic controls grown on iron-sufficient medium. A putative Over-Accumulator of Fe, pOAF40, was identified that expressed high levels of GFP fluorescence. Our objective was to characterize this mutant for alterations in iron homeostasis. Seed of pOAF40 and the non-mutagenized transgenic control were germinated and plants grown on iron-sufficient medium for 14 days before transferring to iron-sufficient or -deficient media for four days. Fer1 mRNA levels, chlorophyll content, and ferric-chelate reductase activity (an enzyme whose activity increases during iron deficiency) were determined at the point of transfer and again four days after transfer. Fer1 mRNA expression was the same at the time of transfer, but greater relative to transgenic controls regardless of iron concentration 4 days later. The average concentration of chlorophyll in pOAF40 was less than the control regardless of sampling time or iron concentration. pOAF40 exhibited lower reductase activity than control on the day of transfer, however this difference in activity was not detected four days after transfer within iron-sufficient or -deficient treatments. Furthermore, ferric-chelatate reductase activity was greater in iron-deficient than -sufficient media for both mutant and control suggesting normal response to iron-deficiency by pOAF40. Further characterization of this mutant is being performed to determine whether the mutation deregulates ferritin expression or leads to over-accumulation of iron in leaves.MU Monsanto Undergraduate Research Fellowshi

Allantoate amidohydrolase transcript expression is independent of drought tolerance in soybean

Drought is a limiting factor for N2 fixation in soybean [Glycine max (L.) Merr.] thereby resulting in reduced biomass accumulation and yield. Drought-sensitive genotypes accumulate ureides, a product of N2 fixation, during drought stress; however, drought-tolerant genotypes have lower shoot ureide concentrations, which appear to alleviate drought stress on N2 fixation. A key enzyme involved in ureide breakdown in shoots is allantoate amidohydrolase (AAH). It is hypothesized that AAH gene expression in soybean determines shoot ureide concentrations during water-deficit stress and is responsible for the differential sensitivities of the N2-fixation response to drought among soybean genotypes. The objectives were to examine the relationship between AAH transcript levels and shoot ureide concentration and drought tolerance. Drought-tolerant (Jackson) and drought-sensitive (Williams) genotypes were subjected to three water-availability treatments: well-watered control, moderate water-deficit stress, and severe water-deficit stress. Shoot ureide concentrations were examined, in addition to gene expression of AAH and DREB2, a gene expressed during water-deficit stress. As expected, DREB2 expression was detected only during severe water-deficit stress, and shoot ureide concentrations were greatest in the drought-sensitive genotype relative to the drought-tolerant genotype during water-deficit stress. However, expression of AAH transcripts was similar among water treatments and genotypes, indicating that AAH mRNA was not closely associated with drought tolerance. Ureide concentrations in shoots were weakly associated with AAH mRNA levels. These results indicate that AAH expression is probably not associated with the increased ureide catabolism observed in drought-tolerant genotypes, such as Jackson. Further study of AAH at the post-translational and enzymatic levels is warranted in order to dissect the potential role of this gene in drought tolerance

Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines

BACKGROUND: Iron is one of fourteen mineral elements required for proper plant growth and development of soybean (Glycine max L. Merr.). Soybeans grown on calcareous soils, which are prevalent in the upper Midwest of the United States, often exhibit symptoms indicative of iron deficiency chlorosis (IDC). Yield loss has a positive linear correlation with increasing severity of chlorotic symptoms. As soybean is an important agronomic crop, it is essential to understand the genetics and physiology of traits affecting plant yield. Soybean cultivars vary greatly in their ability to respond successfully to iron deficiency stress. Microarray analyses permit the identification of genes and physiological processes involved in soybean's response to iron stress. RESULTS: RNA isolated from the roots of two near isogenic lines, which differ in iron efficiency, PI 548533 (Clark; iron efficient) and PI 547430 (IsoClark; iron inefficient), were compared on a spotted microarray slide containing 9,728 cDNAs from root specific EST libraries. A comparison of RNA transcripts isolated from plants grown under iron limiting hydroponic conditions for two weeks revealed 43 genes as differentially expressed. A single linkage clustering analysis of these 43 genes showed 57% of them possessed high sequence similarity to known stress induced genes. A control experiment comparing plants grown under adequate iron hydroponic conditions showed no differences in gene expression between the two near isogenic lines. Expression levels of a subset of the differentially expressed genes were also compared by real time reverse transcriptase PCR (RT-PCR). The RT-PCR experiments confirmed differential expression between the iron efficient and iron inefficient plants for 9 of 10 randomly chosen genes examined. To gain further insight into the iron physiological status of the plants, the root iron reductase activity was measured in both iron efficient and inefficient genotypes for plants grown under iron sufficient and iron limited conditions. Iron inefficient plants failed to respond to decreased iron availability with increased activity of Fe reductase. CONCLUSION: These experiments have identified genes involved in the soybean iron deficiency chlorosis response under iron deficient conditions. Single linkage cluster analysis suggests iron limited soybeans mount a general stress response as well as a specialized iron deficiency stress response. Root membrane bound reductase capacity is often correlated with iron efficiency. Under iron-limited conditions, the iron efficient plant had high root bound membrane reductase capacity while the iron inefficient plants reductase levels remained low, further limiting iron uptake through the root. Many of the genes up-regulated in the iron inefficient NIL are involved in known stress induced pathways. The most striking response of the iron inefficient genotype to iron deficiency stress was the induction of a profusion of signaling and regulatory genes, presumably in an attempt to establish and maintain cellular homeostasis. Genes were up-regulated that point toward an increased transport of molecules through membranes. Genes associated with reactive oxidative species and an ROS-defensive enzyme were also induced. The up-regulation of genes involved in DNA repair and RNA stability reflect the inhospitable cellular environment resulting from iron deficiency stress. Other genes were induced that are involved in protein and lipid catabolism; perhaps as an effort to maintain carbon flow and scavenge energy. The under-expression of a key glycolitic gene may result in the iron-inefficient genotype being energetically challenged to maintain a stable cellular environment. These experiments have identified candidate genes and processes for further experimentation to increase our understanding of soybeans' response to iron deficiency stress

A European study investigating patterns of transition from home care towards institutional dementia care: the protocol of a RightTimePlaceCare study

<p>Abstract</p> <p>Background</p> <p>Health care policies in many countries aim to enable people with dementia to live in their own homes as long as possible. However, at some point during the disease the needs of a significant number of people with dementia cannot be appropriately met at home and institutional care is required. Evidence as to best practice strategies enabling people with dementia to live at home as long as possible and also identifying the right time to trigger admission to a long-term nursing care facility is therefore urgently required. The current paper presents the rationale and methods of a study generating primary data for best-practice development in the transition from home towards institutional nursing care for people with dementia and their informal caregivers. The study has two main objectives: 1) investigate country-specific factors influencing institutionalization and 2) investigate the circumstances of people with dementia and their informal caregivers in eight European countries. Additionally, data for economic evaluation purposes are being collected.</p> <p>Methods/design</p> <p>This paper describes a prospective study, conducted in eight European countries (Estonia, Finland, France, Germany, Netherlands, Sweden, Spain, United Kingdom). A baseline assessment and follow-up measurement after 3 months will be performed. Two groups of people with dementia and their informal caregivers will be included: 1) newly admitted to institutional long-term nursing care facilities; and 2) receiving professional long-term home care, and being at risk for institutionalization. Data will be collected on outcomes for people with dementia (e.g. quality of life, quality of care), informal caregivers (e.g. caregiver burden, quality of life) and costs (e.g. resource utilization). Statistical analyses consist of descriptive and multivariate regression techniques and cross-country comparisons.</p> <p>Discussion</p> <p>The current study, which is part of a large European project 'RightTimePlaceCare', generates primary data on outcomes and costs of long-term nursing care for people with dementia and their informal caregivers, specifically focusing on the transition from home towards institutional care. Together with data collected in three other work packages, knowledge gathered in this study will be used to inform and empower patients, professionals, policy and related decision makers to manage and improve health and social dementia care services.</p

Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

As mortality rates decline, life expectancy increases, and populations age, non-fatal outcomes of diseases and injuries are becoming a larger component of the global burden of disease. The Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) provides a comprehensive assessment of prevalence, incidence, and years lived with disability (YLDs) for 328 causes in 195 countries and territories from 1990 to 2016

Investigations of iron-regulated genes in Arabidopsis thaliana [abstract]

Abstract only availableIron is essential for virtually all life, but little is known about the way plants sense, take up, and maintain iron. In an effort to learn more about how plants use this vital nutrient, two projects using the model plant Arabidopsis thaliana were undertaken. The first was a novel genetic screen using transgenic plants harboring the reporter Green Fluorescent Protein (GFP) driven by the iron-regulated AtFer1 promoter. One mutant, OAF 102, was identified for its constitutively high GFP fluorescence relative to unmutagenized transgenic controls. OAF 102 has constitutively elevated GFP mRNA but retains iron-dependent expression of AtFer1; all other iron-related traits tested were similar in OAF 102 and the transgenic parent. Sequencing of the transgenic promoter from OAF 102 did not reveal any mutations in the TATA box, G Box, or iron-dependent regulatory sequence (IDRS). The decoupled regulation of these genes may indicate an epigenetic mutation or a mutation elsewhere in the genome. The other project is an investigation of mutant lines singly and doubly mutant for T-DNA insertions in four bHLH transcription factors found to be upregulated during iron deficiency. Neither single nor double mutant lines showed alterations in ferric-chelate reductase activity. This may reflect a role in iron homeostasis that is not detectable using this tool or a degree of functional redundancy. Further investigation of these T-DNA lines and of OAF 102 is ongoing.MU Monsanto Undergraduate Research Fellowshi

Adventures in Arabidopsis: Two projects investigating iron homeostasis in plants [abstract]

Abstract only availableFaculty Mentor: Elizabeth Rogers, BiochemistryIn order to identify genes involved in iron homeostasis, a novel genetic screen was developed.  Seed from a line transgenic for Green Fluorescent Protein (GFP) driven by the Fer1 promoter, a gene whose mRNA expression levels are increased during iron sufficiency and thus reflects iron status in plants were mutagenized with EMS and screened for high or low GFP fluorescence relative to non-mutagenized transgenic controls.  From this screen, approximately 300 putative mutants for iron homeostasis were identified based on either high or low fluorescence relative to the transgenic control on iron-sufficient media.  In order to more accurately determine the GFP protein level in the plants, total protein from each line grown on iron-sufficient media for 14 days then transferred to iron-deficient or fresh iron-sufficient media is being extracted, and will be separated by SDS-PAGE, and probed for GFP using a two-antibody detection system. One mutant, OAF102, had been previously identified for its constitutive fluorescence during both iron-sufficient and -deficient conditions three days after transfer, and had been shown to have uncoupled GFP and Fer1 mRNA levels between iron-sufficient and -deficient media at this point.  OAF 102 and the non-mutagenized transgenic control were grown for 14 days on iron-sufficient media, then transferred to iron-sufficient or -deficient media.  GFP and Fer1 mRNA levels were determined by northern blotting at 2, 3, 4, and 5 days after transfer.  Two repetitions of the experiment were performed.  Neither one showed the uncoupling seen in previous experiments at three days after transfer.  OAF 102 may have constitutively elevated GFP mRNA, but normal regulation of Fer1 mRNA levels.  Results have not been consistent and more experiments are being done.  Four closely related transcription factors, bHLH's 038, 039, 100, and 101, had previously been shown to be upregulated in Arabidopsis during iron deficiency, but T-DNA insertion lines for each transcription factor did not show any changes in ferric-chelate reductase activity, chlorophyll content, or iron content, all of which are indicators or iron status in the plant.  In order to further evaluate the potential role of these transcription factors in iron homeostasis, crosses were performed to create five double mutants with T-DNA insertions in two o