Proteomic characterization of iron deficiency responses in Cucumis sativusL. roots

Iron is an essential element for all living organisms, being part of many proteins participating in fundamental mechanisms such as DNA synthesis, respiration, photosynthesis and metabolism [1]. In plants, the main cause of Fe deficiency is its low availability in the soil solution due to the scarce solubility of its compounds in well aerated environments. To cope with this problem plants have developed efficient mechanisms to acquire Fe from the soil. Two main strategies are known: dicots and non-graminaceous monocots operate applying what is known as Strategy I, while graminaceous monocots operate with the so-called Strategy II [2, 3]. In the last decade a great amount of biochemical and molecular data have been acquired, increasing the knowledge about the mechanisms adopted by Strategy I plants, especially when grown in the absence of Fe. In particular, three main events seem to assure iron uptake. First, the induction of the reducing activity of a Fe³⁺-chelate reductase (FC-R) located at the plasma membrane of epidermal root cells. The enzyme was first cloned in Arabidopsis (AtFRO2) [4] and FRO2 homologues were found in other Strategy I plants [5–7]; second, the induction of a Fe²⁺ transporter belonging to the ZIP family of proteins [8] and identified as IRTs in several plants [9, 10]; third, the activation of a P-type H⁺-ATPase [11–13] necessary to decrease the apoplastic pH, thus favouring, on one hand, the solubilization of external Fe compounds and the activity of the FC-R [14, 15] and, on the other hand, to establish an effective driving force for Fe uptake [11, 16, 17]. Since the maintenance of these activities requires the constant production of energetic substrates, changes in metabolism have also been studied under Fe deficiency conditions. It has been shown that the rate of glycolysis is increased [18, 19]; the pentose phosphate pathway is increased, as well, to produce both reducing equivalents and carbon skeletons [18, 20]. Furthermore, the phosphoenolpyruvate carboxylase (PEPC) activity has been shown to increase several times under Fe deficiency [21, 22]. This enzyme is very important in the economy of the cell, since it can accomplish several tasks: (i), by consuming PEP it increases the rate of glycolysis, releasing the negative allosteric control exerted on phosphofructo kinase-1 (PFK-1) and aldolase by this phosphorylated compound [23]; (ii), it contributes to the intracellular pH-stat mechanisms [24] and (iii), it forms organic acids, in particular malate and citrate, that may play an important role in the transport of iron through the xylem to the leaf mesophyll [25, 26]. Furthermore, PEPC activity sustains the anaplerotic production of carbon skeletons for biosynthetic pathways (in particular the synthesis of amino acids) and along with the accumulation of di-tricarboxylic acid carrier (DTC), increases the communication between the cytosolic and mitochondrial pools of organic acids, to help maintaining a higher turnover of reducing equivalents [27]. Implication of metabolism has been also inferred from the microarray analysis performed on Fe-starved Arabidopsis plants [28], in which it was shown that the levels of several transcripts encoding enzymes of these metabolic pathways were increased. However, the changes in transcript levels are not a direct proof that the encoded proteins have changed, but that relevant metabolic pathway or biological processes have been affected. To study a global change in the concentration of proteins the new proteomic technologies can be undoubtedly of great help. Concerning plant iron nutrition, two recent studies have analysed by 2-DE the proteome of wild-type tomato and its fer mutant [29, 30] grown under Fe deficiency, to identify to what extent the transcription factor FER influences the accumulation of Fe-regulated protein, while another one analysed the changes in proteomic and metabolic profiles occurring in sugar beet root tips in response to Fe deficiency and resupply [31].

Cucumber (Cucumis sativus L.) plants develop rapid responses to Fe deficiency, and previous works by our and other groups have described very important changes, not only in the classical responses of Strategy I plants, i.e. FC-R and H⁺-ATPase activities, but also in the metabolic rearrangement induced by Fe starvation [7, 18, 19, 32, 33].

In this work we have carried out a proteomic analysis on proteins isolated from cucumber roots grown in the presence or in the absence of Fe for 5 and 8 d. Furthermore, we chose to analyse only the cytosolic soluble protein fraction without contaminations by organelles or membranes.

In this work we have analyzed the soluble proteins extracted from cucumber roots grown in the presence or in the absence of Fe at two different dates, 5 d and 8 d, by 2-DE. Recently, some proteomic studies on Fe deficiency responses have appeared in the literature [29–31]. The first two papers reported the differential expression of proteins in two tomato lines: the T3238-FER genotype and its Fe uptake-inefficient mutant T3238-fer. The former [29] was a study addressed to the identification of a diverse set of differentially accumulated proteins under the control of FER and/or Fe supply, while the latter [30] was a study on total root proteins extracted from these two tomato genotypes, with the increase/decrease being evaluated in a single date after one week of treatment. The third paper [31] reports changes in the proteomic profiles of sugar beet root tips in response to Fe deficiency and resupply.

In order to correlate the metabolic evidences so far obtained in roots of Fe-deficient plants, we have restricted our research to the soluble cytosolic proteins in order to avoid any interference by other cellular systems. Furthermore, we have applied another restriction by characterizing only those spots which showed a two-fold increase or decrease. Under these experimental conditions, 44 proteins that change their level of accumulation were identified. Twenty-one out of 44 increased their concentration under Fe deficiency. Among these, the majority (42% of the total) are enzymes belonging to the glycolytic pathway, confirming previous biochemical data suggesting the involvement of metabolism, and in particular of glycolysis, in response to Fe deficiency. In fact, previous biochemical evidences had shown that under these growing conditions the activities of hexokinase (HK), ATP-dependent phosphofructokinase-1 (ATP-PFK1), glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) and pyruvate kinase (PK) were increased [18, 19, 34]. Surprisingly, none of these enzyme was detected in this proteomic study, but other enzymes of this pathway such as PP-dependent phosphofructokinase (PP-PFK), aldolase, phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM) and enolase were detected and found to be enhanced by Fe deficiency. This discrepancy could be explained by several factors. First of all, it is always risky to strictly link protein levels to their activities: these glycolytic enzymes, in fact, are known to be highly regulated by allosteric mechanisms [23]. In our case, it is thus possible that such mechanisms act in concert with slight increases in the amount of proteins, which might be not considered after the statistical analysis for the subsequent MS analysis. The incomplete match between the levels of some glycolytic enzymes and their activities is also supported by gene expression and the microarray analysis conducted on Arabidopsis, that revealed that only ATP-PFK1, PGK, PGM and enolase transcripts increase in Fe-deficient roots after seven days of Fe starvation, while for HK, GAP-DH and PK a decrease was shown, corroborating in some way our proteomic data [28]. Finally, the peculiarities of the electrophoretic approach must be taken into account. For instance, it is possible that some glycolytic enzymes were not considered in this analysis because of the pI or the molecular weight ranges employed, comigration phenomena and problems of saturation staining.

The same major discrepancy occurs for the PEPC activity whose increase was around 4 fold in cucumber roots, but it was not detected in this proteomic study. The same discrepancy was also found in the proteomic study carried out on sugar beet root tips [31]. However, the amount of protein as determined by immunochemical identification indicated a consistent increase after 10 d of Fe starvation, while if we compare the times used in this work the enhancement between the control and -Fe conditions was less evident [21] and perhaps below the two fold-increase considered for the successive identification by mass spectrometry. Furthermore, the increase in the activity of PEPC could be related to the complex regulation of this enzyme exerted by the positive allosteric effector Glucose-6-P, whose level has been shown to increase under Fe deficiency [19], and by post-translational regulation [40]. These data are in agreement with the microarray analysis [28] done in Arabidopsis, which shows that the PEPC transcript increase occurs only at the 5th d of Fe deficiency, while at the 7th d the transcript is undetectable. While our data on the glycolytic enzymes are in good agreement with those obtained by Rellán-Álvarez et al [31], they agree only in part with those of Li et al [30], since they found that only enolase and triose-P-isomerase increase their level, while, on the contrary, the aldolase activity decrease; from this point of view our data on the involvement of glycolytic enzymes give a much more complete picture. The increase in the glycolytic pathway under Fe deficiency has been confirmed by many biochemical data obtained by several groups [18, 19, 22] and by the proteomic data described in this work, and is in agreement with the major request of energy, reducing equivalents and carbon skeletons to sustain the greater energetic effort and the request of substrate for the synthesis of the large amount of mRNAs and proteins related to this response [41, 42]. Another interesting result is the increase of alcohol dehydrogenase (spot numbers 1519 and 1593) that would confirm the involvement of anaerobic metabolism in response to Fe deficiency [22]. This increase is also in agreement with the microarray study in Arabidopsis [28] in which the transcript for the alcohol dehydrogenase was found to be increased.

The metabolic changes induced by Fe deficiency on the protein pattern is not confined only to glycolysis but other pathways seem to be rearranged as a consequence of this stress, as it occurs for instance in the mitochondria [27, 33]. In fact, we found that enzymes related to carbohydrate metabolism might be suppressed or increased. In particular, enzymes related to the biosynthesis of cell wall polysaccharides such as invertase, 1,4-β-xylosidase and UDP-Glucose dehydrogenase (UDP-Glc-DH) are decreased (Table 2). The decrease in the biosynthesis of the cell wall polysaccharides in Fe-deficient roots would mean a decrease in carbon flux towards the synthesis of cell wall (more likely less important in these conditions) favoring instead glycolysis and other biosynthetic pathways. Moreover, the cell wall can be considered, in conditions where the photosynthetic apparatus might be damaged or not properly working, as a temporary source of carbohydrates. In order to sustain this change in metabolism we found an increased concentration of galactokinase after 8 d of Fe deficiency, which would channel carbon skeletons originating from cell wall degradation to fuel glycolysis. This enzyme is involved in the metabolism of D-galactose-containing oligo- and polysaccharides and occurs in various plants. The raffinose family of oligosaccharides (RFOs) ranks next to sucrose in their abundance in plant kingdom [43]. Plant cell wall contains numerous polysaccharides which consist of a wide range of different sugar residues. An analysis of Arabidopsis identified glucose, rhamnose, galactose, xilose, arabinose and galacturonic and glucuronic acids as the major sugar constituent in the cell wall [44], while a study on the changes of metabolites occurring in sugar beet root tips under Fe deficiency showed a large increase in the RFO sugars [31]. Galactokinase belongs to a sugar-1-P kinase family which account for hydrolysis and recycle of pectic polymers. RFOs might therefore be an important source of rapidly metabolisable carbon other than function as antioxidant [31], (ROS detoxification has been observed in Fe-deficient roots [45]), then, the increase in RFO could help to alleviate ROS damage produced under Fe deficiency. The simultaneous decrease in enzymes involved in the cell wall synthesis might bring to the observed stunting growth of roots under Fe deficiency. Changes in cell wall metabolism has been also observed in Fe-deficient tomato roots [30] and the decrease in invertase activity could, as suggested by Li et al. [30] decrease the relative level of fructose and explain why a down regulation of fructose metabolism was found in these roots.

Another important group of proteins which increase under Fe deficiency is related to nitrogen metabolism (24%). S-adenosylmethionine synthase, alanine aminotransferase, glutamine synthase 1 (the root isoform of GS) and a C-N hydrolase family protein belong to this group. Concerning this group only the S-adenosylmethionine synthase shows a temporal increase, which is limited to the first date of Fe deficiency (Figure 6). This enzyme is involved not only in the biosynthesis of nicotianamine and phytosiderophores of the mugineic acid family [38], but also in the biosynthesis of ethylene, which has been reported to influence the response of Strategy I plants to Fe deficiency [7]. The other three proteins increase at both dates considered. Among them, the most interesting is the C-N hydrolase family protein. In fact, this family of protein includes several enzymes that are involved in nitrogen metabolism and that cleave nitriles as well as amides. Utilization of these nitrogen compounds usually involves several reduction steps. The final step is the assimilation of NH₄⁺ or its transfer to various intermediates such as keto acids [46]. It is well known that Fe deficiency leads to an increase in the organic acid level which play different roles one of which is linked to the synthesis of amino acids [25]. Our study also shows a decrease in the cytoskeleton proteins actin and tubulin along with the storage protein globulin (Table 2 and Figure 7). An intriguing hypothesis we can drive from these results is that all these proteins might be recycled under Fe deficiency and used as a source of amino acids, carbon skeletons and nitrogen. This could be in agreement with the increase in the C-N hydrolase protein family and, even if with contrasting results, with changes in two spots identified as protein PDIs. PDIs catalyses the rearrangement of disulfide bridges of proteins [47] and in Arabidopsis these family of proteins is encoded by 12 genes [48]. While spot number 858 (Table 1 and Figure 6) increases, the other one, spot number 871 (Table 2 and Figure 7) decreases, especially after 8 d. Contrasting results have been found also for spots identified as heat shock proteins, where in one case (spot number 724) we found an increase while in two cases (spot numbers 757 and 758), on the contrary, a decrease was observed. PDIs and HSP70 are involved in the mechanism(s) of protein folding as molecular chaperones (HSP70) and protein folding catalysts (PDIs) so assuring a proper fold of nascent polypeptides into functional proteins. This variability could be associated with a change in the ratio between biosynthesis an degradation of proteins that could bring to a release of amino acids that might serve both as nitrogen and carbon sources. We are aware that the hypothesis is speculative, but the data obtained in this proteomic study support it. Furthermore, other data obtained in our laboratory (manuscript in preparation) show a decrease in the activity of enzymes of the nitrogen assimilatory pathway, since some of them, such as nitrate reductase and nitrite reductase, are Fe-dependent.

In conclusion, the data obtained in this proteomic profiling study confirm some metabolic changes occurring as a response to Fe deficiency. In particular, our data support the increase in the glycolytic flux and in the anaerobic metabolism to sustain the energetic effort Fe-deficient plants must undertake. In fact, Fe deficiency leads to an impairment of the mitochondrial respiratory chain, so the cell must overcome this problem by activating alternative pathways to sustain the energetic requirement and the NAD(P)H turnover [33, 49]. We also found a decrease in the amount of enzymes linked to the biosynthesis of complex carbohydrates of the cell wall, and, on the other hand, an increase in enzymes linked to the turnover of proteins. In a scenario in which the production of new carbon skeletons is strongly impaired by a less efficient photosynthetic apparatus, the plant must face the increased demand of energy and organic compounds. This "cellular effort" seems to be comparable with that occurring in the mammalian muscles in which a strong energetic effort, caused by an enhanced muscular activity, stimulate the anaerobic pathway to produce energy [27]. In Fe-deficient plants, the effort is much more complex, since the contribution of photosynthesis is poor and the plant must recover carbon skeletons from other sources to sustain metabolism. We are aware that more work is necessary to better understand what is going on under Fe deficiency, but the data obtained in the present proteomic work along with those on metabolic activities could cast new light on the responses induced by Fe-deficient plants.