Transcriptomic and proteomic studies in Fe deficient plants have also reported increases in root transcript and protein abundances, respectively, of enzymes related to the glycolytic and TCA cycle pathways, among others. Iron deficiency also induces an accumulation of organic acids, mainly malate and citrate, in roots. The induction of C metabolism in roots of Fe-deficient plants would not only provide a source of reducing power, protons and ATP for the Fe reductase and H+-ATPase enzymes, but also lead to an anaplerotic root C fixation. Carbon accumulated in roots is exported in the form of organic acids via xylem to leaves, which have otherwise drastically reduced photosynthetic rates when Fe-deficient. The higher energy requirements in Fedeficient root cells are tackled by increasing mitochondrial oxidative processes, and roots from Fe-deficient plants show enhanced respiratory activities and higher O2 consumption rates. On the other hand, the mitochondrial respiratory chain is strongly affected under Fe-deficient conditions, since some of its components are Fe-containing enzymes.Furthermore, an increase in anaerobic metabolism has also been described in Fe-deficient roots,hydroponic vertical farm probably as an strategy to oxidize all the reducing power generated by glycolysis and TCA cycle that can not be easily oxidized in the respiratory chain.
When resupplied with Fe, Fe-deficient plants reorganize its metabolism by readjusting metabolic cycles and changing root morphology towards those typical of Fe-sufficient plants.The most common approach used to study Fe deficiency in roots is to analyze only a small number of genes, proteins and/or metabolites. A more comprehensive knowledge of the processes taking place in Fe-deficient roots has been recently provided by the application of modern techniques allowing for the simultaneous and untargeted analysis of multiple genes or proteins. The aim of this work was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply, in order to provide a holistic view of the metabolic processes occurring in plants under different Fe status.The polypeptide pattern of root tip extracts was obtained by 2-D IEF-SDS PAGE electrophoresis. Real scans of typical 2-D gels are shown in Figure 1; an average number of 141 and 148 polypeptides were detected in Fe-sufficient and Fe-deficient plants, respectively . The total number of spots detected was relatively low when compared to other proteomic studies. Several causes may account for this discrepancy, including i) protein extraction method and amount of protein loaded in the gels, ii) gel size, iii) pI range and iv) sensitivity of the staining method. Averaged 2-D polypeptide maps were obtained using gels of three independent preparations, each from a different batch of plants . To better describe polypeptide changes we built a composite averaged virtual map containing all spots present in both Fe-deficient and control root tip extracts . Iron deficiency caused 2-fold increases in 29 spots and 2-fold decreases in signal intensity in 13 spots . Furthermore, 6 spots were only detected in Fe-sufficient plant samples and 13 spots were only detected in Fe-deficient plants .
All polypeptides in the composite averaged map are depicted again in Figure 1D, to permit annotation of those polypeptides where identification was achieved by matrix assisted laser desorption ionization – time of flight MS . These polypeptides were labeled from a to v as described in Figure 1D, and homologies found are described in detail in Table 1. From the 29 spots that showed increases in signal in root tip extracts of Fe-deficient as compared to Fe-sufficient controls, the 20 more abundant were excised and analyzed by MALDI-MS. Since the sugar beet genome has not been sequenced yet and few sequences are avail-able in the databases, identification was performed by homology searches with proteins from other plant species. From the 20 spots analyzed, 14 proteins were identified . These include proteins related to glycolysis such as fructose 1,6-bisphosphate aldolase , triose-phosphate isomerase , 3-phosphoglycerate kinase and enolase . Three spots gave significant matches to malate dehydrogenase , and two more polypeptides presented homology with α and β subunits from F1 ATP synthase . Other proteins increasing in root tip extracts from Fe-deficient sugar beet plants as compared to the controls were fructokinase and formate dehydrogenase . Also, one spot gave significant matches to a cytosolic peptidase At1g79210/YUP8H12R_1 . Spot n gave significant match to a glycine rich protein, which possibly has a role in RNA transcription or processing during stress conditions. From the 13 spots detected de novo in proteome maps from root tip extracts of Fe-deficient plants , the 6 more abundant were excised and analyzed by MALDI-MS, resulting in only 2 positive matches . These significant matches were found for glyceraldehyde 3-phosphate dehydrogenase and DMRL .Changes in the amount of DMRL as well as DMRL gene expression and flavin analysis were further studied using root tip extracts of Fe-sufficient, Fe-deficient and Feresupplied sugar beet plants .
From the 13 spots showing a decrease in signal intensity in root tip extracts from Fe-deficient plants as compared to controls , 3 were identified by MALDI-MS. Spots q and r gave a significant match to nucleoside diphosphate kinase I and to oxalate oxidase-like germin, respectively. 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 .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. Iron deficiency caused significant changes in the response ratios of 26 metabolites. 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 farm 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 [9,28]. On the other hand, these compounds are secreted to the growth media at low pH [6] 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 , 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 microarray 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.