Spot s presented homology with the At4g27270 protein whose molecular function is to interact selectively with FMN, and also presents oxidoreductase activity. From the 6 spots not detected in root tip extracts from Fe-deficient plants as compared to the controls , 3 were identified by MALDI-MS . Proteins matched were oxalate oxidase , peroxidase and caffeoyl CoA Ometyltransferase .Changes induced by Fe-deficiency and Fe-resupply in the root tip metabolome were evaluated by non-biased gas chromatography mass spectrometry metabolite profiling. A total of 326 metabolites were present in at least 80% of the samples of at least one treatment, and 77 of them were identified. Partial least square analysis shows a good separation between +Fe and -Fe root tips . Iron-deficient samples were closer to the 24 h and 72 h YZ samples than to the 72 h WZ ones. On the other hand, the 72 h WZ samples were closer to the +Fe samples than to the -Fe ones.Iron deficiency and/or resupply caused significant changes in the levels of 62 out of the 77 identified metabolites. Metabolite level data were normalized to the mean response of the +Fe treatment; response ratios, defined as the level in a given treatment divided by the level in the +Fe control, are indicated in Table 2.Large increases were found for some organic acids , some sugars , nicotianamine and 2-aminoadipic acid. The response ratio of oxalic acid decreased markedly in -Fe conditions, whereas those of other aminoacids, N compounds, lipid metabolites and others did not show large changes when compared to the Fe-sufficient controls.
Twenty-four hours after Fe-resupply, there was a dramatic coordinated increase in the root tip response ratios of galactinol, raffinose, lactobionic acid, cellobiose and nicotianamine when compared to those found in Fe-deficient roots,vertical aeroponic tower garden whereas the response ratios of sucrose, myoinositol, citrate and malate decreased. Seventy-two hours after Fe resupply, the response ratios of galactinol, raffinose, cellobiose, nicotianamine and many other compounds had decreased in the YZ areas, whereas in the WZ the response ratios were very low. The response ratio of many of the lipids increased moderately in all Fe resupplied samples. Metabolites in the coenzyme, glycolysis, oxidative stress, pentose phosphate pathway and signalling categories did not show large response ratio changes with Fe resupply.The changes induced by Fe deficiency in the root tip proteome and metabolome from sugar beet plants grown in hydroponics have been studied. More than 140 proteins and 300 metabolites were resolved in sugar beet root tip extracts. Iron deficiency resulted in significant and higher than 2-fold changes in the relative amounts of 61 polypeptides, and 22 of them were identified. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. In general, our results are in agreement with previous transcriptomic, proteomic and enzymatic studies on Fe-deficient roots. Our data confirm the increases previously found in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways. Two major changes induced by Fe deficiency in roots are described in this study for the first time: the increase in DMRL synthase protein concentration and gene expression, and the increase in RFO sugars. The largest change found in the proteome map of root tip extracts from sugar beet plants grown in Fe deficiency conditions corresponded to DMRL synthase, which was detected de novo in Fe-deficient root tips, and is the protein with the highest concentration in these gels . This enzyme catalyses the fourth step of Rbfl biosynthesis, and Rbfl is the precursor of Rbfl sulphates, FMN and FAD, the last one being a cofactor for the root plasma membrane Fe reductase.
The expression of BvDMRL decreased drastically 24 h after the addition of Fe to Fe-deficient plants, whereas DMRL synthase protein abundance and Rbfl and Rbfl sulphate concentrations did not change significantly with Fe-resupply in the YZ of root tips , suggesting that the turnover of this protein is slow. Accumulation in Fe-deficient roots of flavin compounds, including Rbfl and Rbfl 3′ – and 5′-sulphate is a characteristic response of sugar beet and other plant species. The exact role of flavins in Fe deficiency is unknown, and it has been hypothesized, based on the similar location of flavin accumulation and Fe reduction and on the fact that the Fe reductase is a flavin-containing protein, that free flavin accumulation may be an integral part of the Fe-reducing system in roots from Strategy I plants. On the other hand, these compounds are secreted to the growth media at low pH and, assuming high concentrations at the secretion site, they could mediate extracellular electron transfer between soil Fe deposits and root Fe reductase as it has been described for flavin phosphates secreted by some bacteria. Moreover, excreted flavins could also act as a plant-generated signal that could influence rhizosphere microbial populations, indirectly affecting Fe availability. A major change in carbohydrate metabolism was the large increase in RFO compounds that occurs in roots with Fe deficiency. This increase was higher than that found for sucrose . The total concentrations of raffinose and galactinol were also determined by HPLC-MS, and concentrations of both compounds in the 35-80 nmol g FW-1 range were found in Fe-deficient and Fe-resupplied root tips , whereas concentrations in the +Fe treatment were one order of magnitude lower. The sum of the raffinose and galactinol concentrations in the -Fe, 24h, 72hWZ, 72hYZ and +Fe tissues was 13.9, 7.4, 2.2, 5.1 and 0.6% of the total sucrose, respectively, supporting the relevance of the RFOs changes with Fe status. RFOs have diverse roles in plants, including transport and storage of C and acting as compatible solutes for protection in abiotic stresses .
Other explanationfor the large increase in the relative amounts of RFOs could be the ability to function as antioxidants; galactinol and raffinose have hydroxyl radical scavenging activities similar to other soluble antioxidants such as glutathione and ascorbic acid. Since ROS damage and ROS detoxification strategies have been observed in Fe-deficient roots, the increase in RFO concentration could help to alleviate ROS damage produced under Fe deficiency. Moderate increases in sugars commonly found in cell walls such as cellobiose, xylonic acid and arabinose, which may indicate cell wall modifications, were measured in sugar beet Fe-deficient root tips. Changes in cell wall metabolism have been also described in Fe-deficient tomato roots. On the other hand, it has been described that cell wall damage generates oligosaccharides that can act as signalling molecules in stresses such as wounding. The increase in RFOs could also act as a long distance Fe-deficiency signal via phloem sap transport. This is the first description of RFOs accumulation in plants under Fe deficiency, and the physiological implications of this increase deserve further consideration. Most of the proteins found to be up-accumulated in sugar beet root tips by Fe deficiency were identified as carbohydrate catabolism enzymes, including 5 of the 10 glycolytic pathway enzymes ,vertical gardening in greenhouse one of the citric acid cycle and fructokinase. Increases in the activities and concentrations of several glycolytic enzymes in root extracts with Fe deficiency have been previously found, including fructose 1,6-bisphosphate aldolase, enolase, triosephosphate isomerase and GADPH. Also, increases in the activities and concentrations of several enzymes of the citric acid cycle with Fe deficiency have been previously reported in root extracts, including MDH. Results are also in agreement with micro-array gene analysis in Fe-deficient A. thaliana roots. Increases in the amount of PEPC have been found at the protein level, but this enzyme, with a molecular mass of 110 kDa, was not in the range used in our 2-D gels. Up-regulation of carbohydrate catabolism in roots of plants grown in Fe deficient conditions is probably a result of an increased demand of energy and reducing power in roots needed to sustain the increased activity of H+-ATPase and Fe reductase. Also, two spots corresponding to different subunits of F1 ATP synthase increased in 2-D gels from Fe deficient root tips, further supporting the higher energy requirement in these roots. Moreover, our results show an increase in the amount of formate dehydrogenase, an enzyme related to the anaerobic respiration, in Fe-deficient roots, confirming the results of enzyme and transcriptional analysis. Anaerobic respiration is an alternative pathway for energy production when oxidative phosphorylation is impaired. Metabolite studies revealed large increases in organic acids, including a 20-fold citric acid increase. These increases in TCA cycle organic acids with Fe deficiency are coupled with increases in glycolysis and root C fixation by PEPC, and provide an anaplerotic, non-autotrophic C source for leaves which have otherwise reduced photosynthetic rates.
Malate and citrate could also be pumped from the cytosol to the mitochondria via a di-tricarboxylate carrier where they would allow a higher turnover of reducing equivalents. A significant decrease in oxalic acid concentration was observed in Fe deficient root tips, and similar decreases have been reported in Fe-deficient tomato roots. The implications of oxalate concentration decreases with Fe deficiency are still not known, since the role of oxalic acid in plants is quite different from that of the other organic acids, and for a long time it has been considered as a toxin or a metabolic end product . Regarding N and amino-acid compounds, a large increase was measured for nicotianamine, which has been described to play a role in cytosolic Fe availability. A comprehensive representation of the metabolomic and proteomic changes taking place in root tips under Fe deficiency and resupply is shown in Figure 4. Red and yellow symbols indicate major and moderate increases in metabolites and proteins compared to the Fe-sufficient controls. Blue and green symbols indicate major and moderate decreases in metabolites and proteins compared to the controls. Besides the major increases in RFOs and DMRL, Fe deficiency induced significant changes in root tip metabolism, mainly associated to increases in carbohydrate catabolism, glycolysis and TCA cycle and to a lesser extent in aminoacid and nitrogen metabolism . Similar changes were observed in the 24 and 72h YZ Fere supplied roots, whereas the WZ of 72 h Fe-resupplied plants did not show major changes when compared to +Fe plants . On the other hand, the relative amount of lipid metabolism compounds did not change markedly in Fe-deficient roots, whereas Fe resupply caused a moderate increase in this type of metabolites .Sugar beet was grown as described elsewhere. “Monohil” was always used, with the exception of raffinose and galactinol analysis, which was carried out with “Orbis”. After seed germination in vermiculite and 2 weeks in half-strength Hoagland’s nutrient solution with 45 μM Fe-EDTA, plants were transferred into 20 L plastic buckets containing half strength Hoagland’s nutrient solution with either 0 or 45 μM Fe-EDTA. The pH of the Fe-free nutrient solution was buffered at approximately 7.7 by adding 1 mM NaOH and 1 g L-1 of CaCO3. In the Fe resupply experiments, plants grown for 10 d in the absence of Fe were transferred to 20 L plastic buckets containing half strength Hoagland’s nutrient solution, pH 5.5, with 45 μM Fe-EDTA. The root sub-apical region from Fe-sufficient plants , Fe-deficient plants , Fe-deficient plants resupplied with Fe for 24 h and Fe-deficient plants resupplied with Fe for 72 h was collected with a razor blade and immediately frozen in liquid N2. The specific regions of root sampled were: in the case of +Fe, -Fe and 24 h plants, the first 10 mm from the root apex ; in the case of 72 h Fe resupplied roots two zones were sampled separately, the first 5 mm from the root apex, where a new white zone had developed , and the next 5 mm, comprising the still swollen and yellow root zone . Samples were taken at approximately 4 h after light onset in the growth chamber.Protein extracts were obtained as described elsewhere and protein concentration was measured with RC DC Protein Assay . A first dimension isoelectric focusing separation was carried out on ReadyStrip IPG Strips , using a linear pI gradient 5-8. Strips were loaded in a PROTEAN IEF Cell and focused at 20°C, for a total of 14000 V.h. For the second dimension polyacrylamide gel electrophoresis , IPG strips were placed onto 12% SDS-PAGE gels to separate proteins between 10 and 100 kDa. Proteins were stained with Coomassie-Blue R- 250 and results analyzed with the PDQuest 8.0 software.