Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic analysis of PAA short sequence-dependent regulation of bHLH104 in Arabidopsis thaliana iron homeostasis

ABSTRACT: Plants need iron but excess is harmful. Iron uptake transcription factor bHLH104 is negatively controlled by Fe-binding E3 ligase BTS. We show that three–amino acid terminal sequence of bHLH104 controls its breakdown by the BTS pathway. Removing PAA keeps bHLH104 active, driving constant iron uptake and iron accumulation phenotypes. Targeting this motif can be a strategy for crop biofortification. RNA-seq was performed on Col-0 and transgenic Arabidopsis lines expressing wild-type bHLH104, bHLH104 lacking the PAA short sequence(bHLH104dPAA). Two developmental stages were analyzed: 10-day-old seedlings (shoots and roots) and 30-day-old reproductive stage plants (young leaves, old leaves, roots). Plants were grown under iron-sufficient (+Fe) or iron-deficient (-Fe) conditions. Poly-A selected, strand-specific libraries were sequenced on Illumina NovaSeq 6000 (2×150 bp, ~8 Gb/sample).

INSTRUMENT(S): Illumina NovaSeq X

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Dibin Baby

PROVIDER: E-MTAB-16555 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Transcriptional profile of whole roots of wild-type and pye-1 mutants exposed to 24 hours -Fe were generated Global population increases and climate change underscore the need for better comprehension of how plants acquire and process nutrients such as iron. A systems biology approach was taken to elucidate novel regulatory mechanisms involved in plant responses to iron deficiency (-Fe). Using cell-type specific transcriptional profiling we identified a pericycle-specific iron deficiency response, and a previously uncharacterized transcription factor, POPEYE (PYE), that plays an important role in this response. Functional analysis of PYE suggests that it positively regulates growth and development under iron deficient conditions. ChIP-on-chip analysis and transcriptional profiling reveal that PYE helps maintain iron homeostasis by directly and indirectly regulating the expression of ferric reductases, metal ion transporters, iron storage proteins, and other key iron homeostasis genes. In addition to PYE, we also identified a second protein BRUTUS (BTS), which appears to negatively regulate the response to iron deficiency. BTS is a unique putative E3 ligase protein, with metal ion binding and DNA binding domains. PYE and BTS are tightly co-regulated and physically interact with PYE paralogs, one of which is thought to positively regulate expression of genes involved in iron homeostasis. We propose that iron content is sensed within the pericycle where PYE, perhaps in conjunction with BTS and other regulatory proteins, is then activated to control a regulatory network involved in maintaining proper iron distribution in plants. Keywords: Expression analysis To determine how loss of PYE expression affects the transcriptional profile of whole roots, pye-1 mutants and wild-type seeds were germinated under standard growth conditions then transferred to standard media (control, MS media) or iron deficient media (-Fe, 0.3mM Ferrozine in MS media containing no ferrous sulfate). After 24 hours of exposure to +Fe or -Fe whole roots were collected and analyzed.

Project description:Essential metals such as iron are required for healthy plant growth. Fe is an important cofactor and catalytic element in many biological processes. Fe and other metals can also be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalisation. The NAS genes are supposed to play an important role in Fe homeostasis. They are coding for enzymes called nicotianaminesynthase (NAS), which synthesize nicotianamine (NA) by a one-step condensation reaction of three molecules S-adenosyl-methionine. NA acts as a chelator for Fe, Cu, Ni and Zn and might be involved in the transport and allocation of Fe throughout the plant. We generated quadruple T-DNA insertion mutant nas plants to investigate NA function as described in Klatte et al., 2009, Plant Physiol. The nas4x-1 plants show an interveinal leaf chlorosis when turning from vegetative to reproductive stage, which intensifies when growing under Fe deficiency conditions. nas4x-1 plants have strongly reduced NA contents and show an elevated Fe deficiency response in roots. By performing microarray experiments we want to reveal global changes on transcriptional level in roots and leaves of nas4x-1 mutant compared to wild type plants grown under Fe supply and Fe deficiency conditions, respectively. The loss of NAS genes has a strong impact on the regulation of other metal homeostasis genes and allows to draw conclusions about nicotianamine function in metal homeostasis of A.thaliana. For this study, four-week old nas4x-1 mutant and wild type plants were exposed for 7 days to plant medium with and without Fe supply. These conditions have been established previously and have resulted in a reproducibly strong interveinal leaf chlorosis of nas4x-1 plants compared to wild type, especially upon Fe deficiency conditions. The experiment was repeated three times in consecutive weeks to obtain three independent biological repetitions. Rosette leaves and roots of five week-old plants were harvested, RNA was isolated and microarray hybridization was performed. 24 Total samples were analyzed. We generated the following pairwise comparisons: WT + Fe vs. – Fe, nas4x-1 + Fe vs. – Fe, + Fe WT vs. + Fe nas4x-1, - Fe WT vs. - Fe nas4x-1, roots and leaves

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Transcriptomic analysis of PAA short sequence-dependent regulation of bHLH104 in Arabidopsis thaliana iron homeostasis

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