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

Abstract

Background: Plants 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.

Results: Root 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.

Conclusions: The 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.

Figure 1

2-D IEF-SDS PAGE proteome maps of root tips from Fe-sufficient and Fe-deficient Beta vulgaris plants. Proteins were separated in the first dimension in linear (pH 5-8) IPG gel strips and in the second dimension in 12% acrylamide vertical gels. Scans of real typical gels of root tips from Fe-sufficient and Fe-deficient plants are shown in A and B, respectively. Virtual composite images, showing all polypeptides present in root tips from Fe-deficient and Fe-sufficient plants are shown in panels C and D. Statistical significance was assessed with a t-test (p < 0.10), and a 2-fold change in spot intensity between treatments was used as a threshold. Only spots that fit both criteria are marked in panel C; spots whose intensities decrease or disappear completely with Fe deficiency were labelled with green and blue marks, respectively, and those increasing with Fe deficiency or only present in Fe-deficient gels were labelled with yellow and red marks, respectively. In panel D, polypeptides that had significant homologies with proteins in the databases (using MALDI-MS and MASCOT) are marked by squares and labelled with lower-case letters. Homologies are described in detail in Table 1.

Figure 2

DMRL and flavin analysis. Semi-quantitative RT-PCR analysis of the BvDMRL and actin gene transcripts (A), zoom scans of typical gels containing the BvDMRL protein (B) and riboflavin sulphates (C) and riboflavin (D) concentrations in sugar beet root tips from Fe sufficient (+Fe), Fe-deficient (-Fe), 24 h Fe-resupplied, 72 h Fe-resupplied YZ and 72 h Fe-resupplied WZ. Letters in (C) and (D) indicate significant differences (n = 6, p < 0.05, Duncan test).

Figure 3

Score scatter plot of identified metabolites. Partial Least Square (PLS) analysis, showing the score scatter plot of PLS vector 1 (t1) vs. PLS vector 2 (t2) of identified metabolites in sugar beet root tips from Fe sufficient (+Fe, green circles), Fe-deficient (-Fe, red circles), 24 h Fe-resupplied (24h, blue squares), 72 h Fe-resupplied YZ (72h YZ, grey squares) and 72 h Fe-resupplied WZ (72h WZ, orange squares).

Figure 4

Changes in metabolic pathways as affected by Fe status. Pathways related to metabolites and proteins were integrated according to the KEGG database. Statistical t-student tests were performed to both protein and metabolite data to show statistically relevant changes between samples. Red squares indicate metabolites showing significant, marked (more than 4-fold) up-accumulation compared to the controls, and yellow ones mean significant, moderate (less than 4-fold) up-accumulation. Blue squares mean significant, marked (more than 4-fold) down-accumulation, and green ones mean significant moderate (less than 4-fold) down-accumulation. The same colour code was used with proteins, represented by circles. Red circles are proteins detected only in Fe-deficient gels, and yellow ones mean significantly up-accumulated proteins using a 2-fold threshold. Decreased (at least 50%) and missing proteins in the Fe-deficient treatment were represented in green and blue respectively. Each number or letter corresponds to those shown in the corresponding protein and metabolite tables (Tables 1 and 2 respectively). Panels correspond to root tip extracts from plants Fe-deficient (A), 24h after Fe resupply (B), and 72h after Fe resupply (YZ in panel C and WZ in panel D).