Iron (Fe) deficiency is a major constraint for plant growth and affects the quality of edible plant parts. To investigate the mechanisms underlying Fe homeostasis in plants, Fe deficiency-induced changes in the protein profile of Arabidopsis (Arabidopsis thaliana) roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on a LTQ-Orbitrap with high-energy collision dissociation. A total of 4,454 proteins were identified with a false discovery rate of less than 1.1%, and 2,882 were reliably quantified.

Iron (Fe) is an essential mineral nutrient which severely affects the growth, yield and nutritional quality of plants if not supplied in sufficient quantities. We here report that a short C-terminal amino acid sequence consensus motif (IRON MAN; IMA) conserved across numerous, highly diverse peptides in angiosperms, is essential for Fe uptake in plants.

Phosphate (P i ) deficiency impairs plant growth and productivity in many agricultural ecosystems, causing severe reductions in crop yield. To uncover novel aspects in acclimation to P i starvation, we investigated the correlation between P i deficiency-induced changes in transcriptome and proteome profiles in Arabidopsis roots. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 13,298 proteins and 24,591 transcripts, subsets of 356 proteins and 3106 mRNAs were differentially expressed during P i deficiency. Most dramatic changes were noticed for genes involved in P i acquisition and in processes that either liberate P i or bypass P i /ATP-consuming metabolic steps, for example during membrane lipid remodeling and glycolytic carbon flux. The concordance between the abundance of mRNA and its encoded protein was generally high for highly up-regulated genes, but the analysis also revealed numerous discordant changes in mRNA/protein pairs, indicative of divergent regulation of transcription and post-transcriptional processes. In particular, a decreased abundance of proteins upon P i deficiency was not closely correlated with changes in the corresponding mRNAs. In several cases, up-regulation of gene activity was observed solely at the protein level, adding novel aspects to key processes in the adaptation to P i deficiency. We conclude that integrated measurement and interpretation of changes in protein and transcript abundance are mandatory for generating a complete inventory of the compo-

Iron (Fe) is an essential mineral nutrient and an important factor for the composition of natural plant communities. Low Fe availability in aerated soils with neutral or alkaline pH has led to the evolution of elaborate mechanisms that extract Fe from the soil solution. In Arabidopsis (), Fe is acquired by an orchestrated strategy that comprises mobilization, chelation, and reduction of Fe prior to its uptake. Here, we show that At3g12900, previously annotated as scopoletin 8-hydroxylase (S8H), participates in Fe acquisition by mediating the biosynthesis of fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin derived from the scopoletin pathway. S8H is highly induced in roots of Fe-deficient plants both at the transcript and protein levels. Mutants defective in the expression of showed increased sensitivity to growth on pH 7.0 media supplemented with an immobile source of Fe and reduced secretion of fraxetin. Transgenic lines overexpressing exhibited an opposite phenotype. Homozygous mutants grown on media with immobilized Fe accumulated significantly more scopolin, the storage form of scopoletin, supporting the designated function of S8H in scopoletin hydroxylation. Fraxetin exhibited Fe-reducing properties in vitro with higher rates being observed at neutral relative to acidic pH. Supplementing the media containing immobile Fe with fraxetin partially rescued the mutants. In natural Arabidopsis accessions differing in their performance on media containing immobilized Fe, the amount of secreted fraxetin was highly correlated with growth and Fe and chlorophyll content, indicating that fraxetin secretion is a decisive factor for calcicole-calcifuge behavior (i.e. the ability/inability to thrive on alkaline soils) of plants.

Due to its ease to donate or accept electrons, iron (Fe) plays a crucial role in respiration and metabolism, including tetrapyrrole synthesis, in virtually all organisms. In plants, Fe is a component of the photosystems and thus essential for photosynthesis. Fe deficiency compromises chlorophyll (Chl) synthesis, leading to interveinal chlorosis in developing leaves and decreased photosynthetic activity. To gain insights into the responses of photosynthetically active cells to Fe deficiency, we conducted transcriptional profiling experiments on leaves from Fe-sufficient and Fe-deficient plants using the RNA-seq technology. As anticipated, genes associated with photosynthesis and tetrapyrrole metabolism were dramatically down-regulated by Fe deficiency. A sophisticated response comprising the down-regulation of HEMA1 and NYC1, which catalyze the first committed step in tetrapyrrole biosynthesis and the conversion of Chl b to Chl a at the commencement of Chl breakdown, respectively, and the up-regulation of CGLD27, which is conserved in plastid-containing organisms and putatively involved in xanthophyll biosynthesis, indicates a carefully orchestrated balance of potentially toxic tetrapyrrole intermediates and functional end products to avoid photo-oxidative damage. Comparing the responses to Fe deficiency in leaves to that in roots confirmed subgroup 1b bHLH transcription factors and POPEYE/BRUTUS as important regulators of Fe homeostasis in both leaf and root cells, and indicated six novel players with putative roles in Fe homeostasis that were highly expressed in leaves and roots and greatly induced by Fe deficiency. The data further revealed down-regulation of organ-specific subsets of genes encoding ribosomal proteins, which may be indicative of a change in ribosomal composition that could bias translation. It is concluded that Fe deficiency causes a massive reorganization of plastid activity, which is adjusting leaf function to the availability of Fe.

BackgroundQuantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants.ResultsHere, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance.ConclusionsThe integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components.

Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms.The removal of introns from the immature mRNA by a process called "pre-mRNA splicing" occurs in the vast majority of eukaryotic protein-coding genes. Pre-mRNA splicing is catalyzed by elaborate ribonucleoprotein megadalton complexes referred to as spliceosomes (Wahl et al., 2009). Multiple mRNA isoforms can be generated from a single gene locus by alternative splicing, potentially producing functionally distinct protein isoforms (Hsu and Hertel, 2009). In mammals, alternative splicing dramatically increases protein diversity, yielding proteins that differ in function, activity, binding properties, or subcellular localization, and is believed to account for the multiexonic gene expression diversity, generating an estimated 100,000 proteins encoded by 25,000 genes in humans (Modrek and Lee, 2002;Kelemen et al., 2013).In humans, more than 95% of the intron-containing genes are alternatively spliced (Pan et al., 2008). Alternative splicing is less prominent in plants, and its importance has been debated. Recent estimates based on RNA sequencing (RNA-seq) data sug...

Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe) storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1). Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140), AtVTL2 (At1g76800) or AtVTL5 (At3g25190) in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.