Jorge Rodríguez-Celma

Mutually Exclusive Alterations in Secondary Metabolism are Critical for the Uptake of Insoluble Iron Compounds by Arabidopsis and Medicago truncatula

Coexpression and promoter analysis under iron deficiency in roots of Arabidopsis and Medicago demonstrates the integral role for production and secretion of compounds that facilitate the uptake of reduction-based iron acquisition. The generally low bioavailability of iron in aerobic soil systems forced plants to evolve sophisticated genetic strategies to improve the acquisition of iron from sparingly soluble and immobile iron pools. To distinguish between conserved and species-dependent components of such strategies, we analyzed iron deficiency-induced changes in the transcriptome of two model species, Arabidopsis (Arabidopsis thaliana) and Medicago truncatula. Transcriptional profiling by RNA sequencing revealed a massive up-regulation of genes coding for enzymes involved in riboflavin biosynthesis in M. truncatula and phenylpropanoid synthesis in Arabidopsis upon iron deficiency. Coexpression and promoter analysis indicated that the synthesis of flavins and phenylpropanoids is tightly linked to and putatively coregulated with other genes encoding proteins involved in iron uptake. We further provide evidence that the production and secretion of phenolic compounds is critical for the uptake of iron from sources with low bioavailability but dispensable under conditions where iron is readily available. In Arabidopsis, homozygous mutations in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase family gene F6′H1 and defects in the expression of PLEIOTROPIC DRUG RESISTANCE9, encoding a putative efflux transporter for products from the phenylpropanoid pathway, compromised iron uptake from an iron source of low bioavailability. Both mutants were partially rescued when grown alongside wild-type Arabidopsis or M. truncatula seedlings, presumably by secreted phenolics and flavins. We concluded that production and secretion of compounds that facilitate the uptake of iron is an essential but poorly understood aspect of the reduction-based iron acquisition strategy, which is likely to contribute substantially to the efficiency of iron uptake in natural conditions.

Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply

BackgroundPlants grown under iron deficiency show different morphological, biochemical and physiological changes. These changes include, among others, the elicitation of different strategies to improve the acquisition of Fe from the rhizosphere, the adjustment of Fe homeostasis processes and a reorganization of carbohydrate metabolism. The application of modern techniques that allow the simultaneous and untargeted analysis of multiple proteins and metabolites can provide insight into multiple processes taking place in plants under Fe deficiency. The objective of this study was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply.ResultsRoot tip extract proteome maps were obtained by 2-D isoelectric focusing polyacrylamide gel electrophoresis, and approximately 140 spots were detected. Iron deficiency resulted in changes in the relative amounts of 61 polypeptides, and 22 of them were identified by mass spectrometry (MS). Metabolites in root tip extracts were analyzed by gas chromatography-MS, and more than 300 metabolites were resolved. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. Iron deficiency induced increases in the relative amounts of proteins and metabolites associated to glycolysis, tri-carboxylic acid cycle and anaerobic respiration, confirming previous studies. Furthermore, a protein not present in Fe-sufficient roots, dimethyl-8-ribityllumazine (DMRL) synthase, was present in high amounts in root tips from Fe-deficient sugar beet plants and gene transcript levels were higher in Fe-deficient root tips. Also, a marked increase in the relative amounts of the raffinose family of oligosaccharides (RFOs) was observed in Fe-deficient plants, and a further increase in these compounds occurred upon short term Fe resupply.ConclusionsThe increases in DMRL synthase and in RFO sugars were the major changes induced by Fe deficiency and resupply in root tips of sugar beet plants. Flavin synthesis could be involved in Fe uptake, whereas RFO sugars could be involved in the alleviation of oxidative stress, C trafficking or cell signalling. Our data also confirm the increase in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways.

Changes induced by two levels of cadmium toxicity in the 2-DE protein profile of tomato roots

Tomato is an important crop from nutritional and economical points of view, and it is grown in greenhouses, where special substrates and the use of recycled water imply an increased risk of Cd accumulation. We investigated tomato root responses to low (10 microM) and high (100 microM) Cd concentrations at the root proteome level. Root extract proteome maps were obtained by 2-DE, and an average of 121, 145 and 93 spots were detected in the 0, 10 and 100 microM Cd treatments, respectively. The low Cd treatment (10 microM) resulted in significant and higher than 2-fold changes in the relative amounts of 36 polypeptides, with 27 of them identified by mass spectrometry, whereas the 100 microM Cd treatment resulted in changes in the relative amounts of 41 polypeptides, with 33 of them being identified. The 2-DE based proteomic approach allowed assessing the main metabolic pathways affected by Cd toxicity. Our results suggests that the 10 microM Cd treatment elicits proteomic responses similar to those observed in Fe deficiency, including activation of the glycolytic pathway, TCA cycle and respiration, whereas the 100 microM Cd treatment responses are more likely due to true Cd toxicity, with a general shutdown of carbon metabolism and increases in stress related and detoxification proteins.

Root Responses of Medicago truncatula Plants Grown in Two Different Iron Deficiency Conditions: Changes in Root Protein Profile and Riboflavin Biosynthesis

Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the worlds agricultural areas, especially those with high soil CaCO(3). In the present work, a two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, including qPCR and riboflavin determination, to investigate Fe-deficiency responses in Medicago truncatula plants grown with and without CaCO(3). Iron deficiency caused a de novo accumulation of DMRLs and GTPcII, proteins involved in riboflavin biosynthesis, as well as marked increases in root riboflavin concentrations and in the expression of four genes from the riboflavin biosynthetic pathway. Two novel changes found were the increased accumulation of proteins related to N recycling and protein catabolism. Other identified changes were consistent with previously found increases in glycolysis, TCA cycle, and stress-related processes. All effects were more marked in the presence of CaCO(3). Our results show that the riboflavin biosynthesis pathway was up-regulated at the genomic, proteomic, and metabolomic levels under both Fe-deficiency treatments, especially in the presence of CaCO(3). Results also indicate that N recycling occurs in M. truncatula upon Fe deficiency, possibly constituting an additional anaplerotic N and C source for the synthesis of secondary metabolites, carboxylates, and others.

The transcriptional response of Arabidopsis leaves to Fe deficiency.

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.

Characterization of Flavins in Roots of Fe-Deficient Strategy I Plants, with a Focus on Medicago truncatula

The root accumulation and excretion of riboflavin (Rbfl) and Rbfl derivatives have been studied in the model legume species Medicago truncatula, grown in hydroponics in two different Fe deficiency conditions, with and without CaCO(3). Using high resolution mass spectrometry techniques coupled to liquid chromatography, three different flavin derivatives not previously reported in plants, putatively identified as 7-hydroxy-Rbfl, 7α-hydroxy-Rbfl and 7-carboxy-Rbfl, were found along with Rbfl in Fe-deficient M. truncatula roots. In the presence of CaCO(3) most of the flavins were accumulated in the roots, whereas in the absence of CaCO(3) there was partial export to the nutrient solution. The major flavins in roots and nutrient solution were Rbfl and 7-hydroxy-Rbfl, respectively. Flavins were located in the root cortex and epidermal cells, preferentially in a root region near the apex that also exhibited increased ferric chelate reductase (FCR) activity. Six out of 15 different species of horticultural interest showed root increases in both Rbfl (four of them also having Rbfl derivatives) and FCR. No significant correlation was found between Rbfl and either phosphoenolpyruvate carboxylase or FCR activities, whereas the latter two showed a good correlation between them. The possible roles of Rbfl and Rbfl derivatives in roots and nutrient solutions are discussed. Medicago truncatula is proposed as a model system for flavin studies.

Time course induction of several key enzymes in Medicago truncatula roots in response to Fe deficiency.

Medicago truncatula constitutes a good model for Strategy I plants, since when this plant is challenged with Fe shortage the most important root physiological responses induced by Fe deficiency are developed, including the yellowing of root tips. A better understanding of the mechanisms involved in root adaptation to Fe deficiency in M. truncatula may strengthen our ability to enhance Fe efficiency responses in other plant species, especially in different agronomically relevant legumes. Riboflavin concentration, phosphoenolpyruvate carboxylase (EC 4.1.1.31) and Fe reductase activities, and acidification capacity have been determined in M. truncatula roots at different time points after imposing Fe deficiency. Root riboflavin concentrations increased with Fe deficiency and concomitantly MtDMRL was upregulated at the transcriptional level, supporting a role for flavins in the Fe deficiency response. Root Fe reductase and phosphoenolpyruvate carboxylase activities as well as acidification capacity were higher in roots of Fe-deficient than in control plants, and the corresponding genes, MtFRO1, MtPEPC1 and MtHA1 were also upregulated by Fe deficiency. Expression of these genes and their corresponding physiological activities followed different patterns over time, suggesting the existence of both transcriptional and post-transcriptional regulation.

The distinct functional roles of the inner and outer chloroplast envelope of Pea (Pisum sativum) as revealed by proteomic approaches.

Protein profiles of inner (IE) and outer (OE) chloroplast envelope membrane preparations from pea were studied using shotgun nLC-MS/MS and two-dimensional electrophoresis, and 589 protein species (NCBI entries) were identified. The relative enrichment of each protein in the IE/OE pair of membranes was used to provide an integrated picture of the chloroplast envelope. From the 546 proteins identified with shotgun, 321 showed a significant differential distribution, with 180 being enriched in IE and 141 in OE. To avoid redundancy and facilitate in silico localization, Arabidopsis homologues were used to obtain a nonredundant list of 409 envelope proteins, with many showing significant OE or IE enrichment. Functional classification reveals that IE is a selective barrier for transport of many metabolites and plays a major role in controlling protein homeostasis, whereas proteins in OE are more heterogeneous and participate in a wide range of processes. Data support that metabolic processes previously described to occur in the envelope such as chlorophyll and tocopherol biosynthesis can be ascribed to the IE, whereas others such as carotenoid or lipid biosynthesis occur in both membranes. Furthermore, results allow empirical assignation to the IE and/or OE of many proteins previously assigned to the bulk chloroplast envelope proteome.

Plant fluid proteomics: Delving into the xylem sap, phloem sap and apoplastic fluid proteomes

The phloem sap, xylem sap and apoplastic fluid play key roles in long and short distance transport of signals and nutrients, and act as a barrier against local and systemic pathogen infection. Among other components, these plant fluids contain proteins which are likely to be important players in their functionalities. However, detailed information about their proteomes is only starting to arise due to the difficulties inherent to the collection methods. This review compiles the proteomic information available to date in these three plant fluids, and compares the proteomes obtained in different plant species in order to shed light into conserved functions in each plant fluid. Inter-species comparisons indicate that all these fluids contain the protein machinery for self-maintenance and defense, including proteins related to cell wall metabolism, pathogen defense, proteolysis, and redox response. These analyses also revealed that proteins may play more relevant roles in signaling in the phloem sap and apoplastic fluid than in the xylem sap. A comparison of the proteomes of the three fluids indicates that although functional categories are somewhat similar, proteins involved are likely to be fluid-specific, except for a small group of proteins present in the three fluids, which may have a universal role, especially in cell wall maintenance and defense. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Changes Induced by Fe Deficiency and Fe Resupply in the Root Protein Profile of a Peach-Almond Hybrid Rootstock

The changes in the root extract protein profile of the Prunus hybrid GF 677 rootstock (P. dulcis × P. persica) grown in hydroponics as affected by Fe deficiency and short-term (24 h) Fe resupply have been studied by 2-dimensional gel electrophoresis-based techniques. A total of 335 spots were consistently found in the gels. Iron deficiency caused above 2-fold increases or >50% decreases in the relative abundance in 10 and 6 spots, respectively, whereas one spot was only detected in Fe-deficient plants. Iron resupply to Fe-deficient plants caused increases and decreases in relative abundance in 15 and 16 spots, respectively, and one more spot was only detected in Fe-resupplied Fe-deficient plants. Ninety-five percent of the proteins changing in relative abundance were identified using nanoliquid chromatography-tandem mass spectrometry. Defense responses against oxidative and general stress accounted for 50% of the changes in Fe-deficient roots. Also, a slight induction of the glycolysis-fermentation pathways was observed in GF 677 roots with Fe deficiency. The root protein profile of 24 h Fe-resupplied plants was similar to that of Fe-deficient plants, indicating that the deactivation of Fe-deficiency metabolic responses is slow. Taken together, our results suggest that the high tolerance of GF 677 rootstock to Fe deficiency may be related to its ability to elicit a sound defense response against both general and oxidative stress.