Fine regulation of leaf iron use efficiency and iron root uptake under limited iron bioavailability

Plant Science 198 (2013) 39–45

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Review

Fine regulation of leaf iron use efficiency and iron root uptake under limited iron bioavailability José M. García-Mina a,b,∗ , Eva Bacaicoa a , Marta Fuentes a , Esther Casanova a a b

R&D Department (CIPAV-Roullier Group) Timac Agro, Polígono Arazuri-Orcoyen c/C, 31160 Orcoyen (Navarra), Spain Chemistry and Soil Chemistry Department, Faculty of Sciences, University of Navarra, Spain

a r t i c l e

i n f o

Article history: Received 30 May 2012 Received in revised form 5 October 2012 Accepted 5 October 2012 Available online 13 October 2012 Keywords: Nutrient use efficiency Non-graminaceous plants Iron use efficiency Iron nutrition Iron deficiency Physiological Fe-stress root responses

a b s t r a c t Numerous studies have investigated the molecular and physiological–morphological mechanisms induced in plant roots in response to specific nutrient deficiencies. Both transcriptional and posttranscriptional mechanisms are involved that increase root uptake under nutrient deficiency. Root nutrient deficiency-stress root responses are mainly regulated by the nutrient status in the shoot. The signals involved in shoot to root cross-talk regulation processes for the activation of nutrient-deficiency induced root responses are not clearly elucidated. The physiological-molecular events in the leaf linked to the nutrient availability for metabolic use, are also poorly known. In this context, we focus our attention on iron plant nutrition. Some experimental evidence suggests the existence of a regulatory system concerned with the optimization of the metabolic use of iron, mainly under conditions of iron starvation. This system seems to be activated by the deficiency in iron-availability for metabolic processes in the leaf and regulates the activation of some iron-stress root responses. This regulation seems to be probably expressed by affecting the production and/or translocation of the activating signal sent from the shoot to the root under conditions of iron deficiency in the shoot. © 2012 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fe-stress root responses in non-graminaceous plants (Strategy I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The control of (Strategy I) physiological Fe-stress root responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental evidence supporting the existence of a system of optimized metabolic use of Fe in the shoot, which co-regulates the activation of (Strategy I) physiological Fe-stress root responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential consequences of the existence of a system to optimize the metabolic use of Fe in the shoot in Fe-fertilization of Strategy-I crops . . . Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction In general, crop physiologists define nutrient-use efficiency combining two factors that are considered as independent to each other: (i) nutrient-uptake efficiency (NupE) that is defined as the ratio between nutrient-uptake by plants and nutrientavailable in soil; and (ii) nutrient-utilization efficiency (NutE) that is the ratio between crop harvested and nutrient-uptake by plant.

∗ Corresponding author at: Timac Agro, R&D Department (CIPAV-Roullier Group), Polígono Arazuri-Orcoyen c/C, 31160 Orcoyen (Navarra), Spain. E-mail address: [email protected] (J.M. García-Mina). 0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2012.10.001

Nutrient-use efficiency (NUE) is obtained by multiplying nutrientuptake efficiency and nutrient-utilization efficiency, thus giving the ratio between grain yield (or fruit, aerial part . . .) and nutrient available in soil [1]: NUE = NupE × NutE = Yield : NavS (nutrient available in soil) These parameters can be applied to all mineral nutrients included in plant nutrition and fertilization [1]. This approach, both involving plant and fertilizer, may be called standard model (Fig. 1A). However this model, although useful, has some conceptual problems.

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Fig. 1. Different models to explain the shoot-root regulation of nutrient-stress root responses (A–B) and iron-stress root responses (C) in non-graminaceous plants. (A) Standard model for the regulation of nutrient-stress root responses: (1) decrease in leaf-nutrient concentration; (2) promotion of nutrient-stress root responses; (3) increase in nutrient root uptake from the rhizosphere; (4) increase in nutrient concentration in the xylem and shoot. (B) Coordinated model for the regulation of nutrient-stress root responses: (1) decrease in the concentration of nutrient available for metabolic processes in leaf; (2) activation of a regulatory network to optimize nutrient-metabolic use in the shoot; (3) if it is not enough, activation of nutrient-stress root responses; (4) increase in nutrient root uptake from the rhizosphere; (5) increase in nutrient concentration in the xylem and shoot. (C) Main phytoregulators involved in the proposed mechanisms for the shoot to root regulation of Fe-stress root responses included in coordinated model: (1) decrease in the concentration of iron available for metabolic processes in leaf; (2) activation of a regulatory network to optimize iron-metabolic use in the shoot; (3–4) if it is not enough, increase of IAA content in shoot and roots; (5) activation of iron-stress root responses influenced by the phytoregulators studied; (6) increase in iron concentration in the xylem and shoot. Abbreviations: Nut: nutrient; Fe: iron; IAA: indole-3-acetic acid; NO: nitric oxide; CKs: cytokinins; JA: jasmonic acid. Increase; Decrease;  Translocation;

Activated process;

Blocked process.

From the definition of NUE it becomes clear that, in fact, this approach does not explicitly consider the possible importance of the concentration of nutrient taken up by the plant, and its relationships with a potential specific regulatory network oriented to optimize the metabolic use of the nutrient present within the plant. In fact, this approach considers that the concentration of nutrient in the shoot is directly related to the efficiency of mechanisms of nutrient uptake in the root. That means that the concept of nutrientefficient plant variety is directly ascribed to the efficiency of root nutrient-uptake mechanisms of this variety (Fig. 1A). As a consequence, this definition of NUE becomes rather unspecific. Thus, a same value of NUE can correspond to different values of NupE and NutE. These conceptual problems might be solved by considering the hypothesis that NupE and NutE are two processes closely related to each other and interacting during plant cycle. A plant that is very efficient in the optimization of the utilization of the nutrient and covers its metabolic needs would need to take up less nutrient amount than a less efficient plant, and produce the same yield. This suggests a hypothesis where by plants might have a specific system that is activated under limitation of nutrient availability in the shoot for metabolic processes that optimizes the metabolic use of

this nutrient. This specific system would be expressed before the activation of the system present in the root that can increase both nutrient root uptake and nutrient availability in the rhizosphere. In this way, the two mechanisms would be co-regulated, and the mechanism in root will be activated when the mechanism in shoot, related to the optimization of nutrient metabolic efficiency, does not cover plant needs. The model derived from this hypothesis is named coordinated model (Fig. 1B). It becomes clear that the key-step that differentiates coordinated and standard models is the presence of an intermediate system responsible for the optimization of nutrient efficiency in the shoot. This system would be functionally placed between the event triggered by the reduction of nutrient concentration in the shoot available for metabolic use, associated with a reduction in nutrient in the xylem and the leaf (leaf-nutrient), and the system of specific responses in the root that increases the fraction of available nutrient in the rhizosphere and nutrient root uptake (Fig. 1A and B). The aim of this review is to study the hypothesis involved in the concept of the coordinated model by discussing the experimental evidence that supports this model in the regulation of iron (Fe) shoot deficiency and Fe root uptake under Fe limiting conditions in non-graminaceous plants.

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2. Fe-stress root responses in non-graminaceous plants (Strategy I)

carried out in plants growing in the presence of different levels of available Fe in the nutrient solution [2].

Iron is a micronutrient that plays important roles in the development of several physiological processes within the plant [2]. Among these processes, those related to photosynthesis have a special relevance due to their direct impact on plant growth and crop yields and quality [2]. However, the potential Fe bioavailability in soil is influenced by many factors associated with the chemical nature of Fe. In alkaline soils, Fe forms water-insoluble hydroxides and oxides, and/or Fe carbonates–bicarbonates, which very significantly decrease Fe bioavailability [2]. Plants then develop Fe deficiency that is expressed, among other symptoms, as a gradual loss of chlorophyll in leaves associated with an intense nervaturelocated greening and inter-nervature yellow (chlorotic) area. The incidence of Fe deficiency affects both fruit-crop yields and quality [2]. Several Fe fertilizers have been developed, either synthetic or natural, with the aim of correcting Fe deficiency in all types of crops, but principally fruit trees [2]. Plants have evolved specific root responses that increase both Fe root uptake ability and the fraction of available Fe in rhizosphere [3]. These responses, both physiological and morphological, are configured differently depending on plant species [3]. Non-graminaceous plants have a specific mechanism, Strategy I, consisting of the activation of Fe(III) reduction in root cells by the action of a Fe(III)-chelate reductase. This activation is coupled with a concomitant activation of the biosynthesis of a Fe(II) transporter [3]. Graminaceous plants utilize another different, specific, whole strategy (Strategy II) consisting of the biosynthesis and release to the rhizosphere of specific organic molecules with Fe chelating ability (phytosiderophores) [3]. This is coupled with the biosynthesis of Fe(III)-phytosiderophore complex root transporters [3]. In this study we will focus our attention on Strategy I plant responses under Fe deficiency. This is because the experimental evidence that we have is related to Strategy I-plants. However, it is very plausible that this intermediate mechanism concerning the optimization of Fe metabolic use in the plant is also present in Strategy II-plants. Non-graminaceous plants have complementary physiological mechanisms in addition to Fe(III) reducing activity, such as rhizosphere acidification by the activation of H+ -ATPases in the plasma membrane, and the release of phenolic compounds with some Fe-chelating-reducing activity, which increase the fraction of Fe(II) in the rhizosphere [2–4]. These physiological responses are normally coupled with morphological changes in root architecture, such as the development of subapical root hairs and transfer cells [2,4]. The molecular basis that regulates these responses has been extensively studied in plant models such as arabidopsis, tomato and cucumber [2,3]. Regarding physiological Fe-stress root responses, arabidopsis has genes that encode Fe(III)-chelate reductase (AtFRO2) and Fe(II) transporter (AtIRT1), which are co-regulated under Fe starvation by a transcription factor named FIT (FER-like Fe-deficiency induced transcription factor) in epidermal tissues (FIT regulatory network) [3,5]. The FIT transcription factor seems to work coupled to bHLHfamily genes forming active heterodimers [3,6]. A new bHLH transcription factor (POPEYE; PYE), related to the regulation of the expression of specific genes under Fe deficiency (including metal transporters and plasma membrane ferric chelate reductase) has been described in the vasculature of arabidopsis [3,7]. This regulatory network could be complementary of FIT regulatory network.

(i) Physiological Fe-deficiency responses were induced in potato roots excised from shoots, indicating that roots alone can respond to Fe-deficiency [8,9]. (ii) The application of foliar Fe (the presence of Fe available for metabolic processes in the shoot) deactivates these responses in roots growing without Fe in the nutrient solution [8,10]. (iii) The availability of Fe for metabolic processes in the shoot governs the activation of physiological Fe-stress responses in the root [10]: in experiments where the root has been divided in two areas, one without Fe and another one with Fe, the (+Fe) root area is able to develop some physiological Fe-stress root responses [10]. This fact indicates that there exists a main regulatory system in the shoot that is able to activate physiological Fe-stress responses in the root under conditions in which the local root Fe-sensing system is deactivated. (iv) In principle, from (ii) we can deduce that there could be a shoot to root repressing signal that deactivates Fe-stress responses in roots cultivated without Fe but without symptoms of Fe deficiency in the shoot. From (iii) we can deduce that there could be an activating shoot to root signal that activates Fe stress responses in roots cultivated in the presence of Fe, but with symptoms of Fe deficiency in the shoot. This result also indicates that shoot to root activating signal is predominant and, therefore, must be able to activate Fe-stress root responses even under conditions of Fe sufficiency. In this context, the work of Enomoto et al. [11], is especially relevant. These authors studied in tobacco roots the expression of genes encoding Fe(III)-chelate reductase (NtFRO1) and Fe(II) transporter (NtIRT1) in plants whose leaves were excised in various patterns. The authors concluded that a shoot to root positiveactivating signal is involved in the regulation of these Fe-stress root responses under Fe starvation. This study confirmed the ability of roots separated from the shoot to respond to Fe starvation, thus indicating the presence of a local Fe-sensing system [11]. However, this study also reported that there was no experimental evidence supporting the shoot to root repressing signal hypothesis [11]. The authors suggest that the shoot to root activating signal is probably much more intense and functionally relevant than the local-root activating signal [11].

3. The control of (Strategy I) physiological Fe-stress root responses The following main facts can be enunciated from the analysis of the results reported by diverse classical and split-root experiments

Specific phytoregulators could play an important role controlling the molecular network responsible for the regulation of this complex system [2,3]. Several studies have reported results supporting an important role of indole-3-acetic acid (IAA) in the activating signal sent by the shoot to the root in plants with Fe deficiency in the shoot. The only hormone that varied significantly under Fe deficiency in cucumber shoot was IAA [12]. The Fe deficiency-induced increase in shoot IAA was correlated to a prompt increase of IAA concentration in the root [12]. Likewise, Fe deficiency was associated with an increase in IAA in the root, in other plant species [13,14]. Specific inhibitors of IAA shoot to root transport and functionality blocked the transcriptional and post-transcriptional activation of some Festress root responses (Fe(III)-chelate reductase/CsFRO1 and Fe(II) transporter/CsIRT1) in cucumber [15]. IAA was also able to activate physiological Fe-stress root responses at transcriptional and post-transcriptional levels in cucumber under conditions of Fesufficiency in the root and without symptoms of Fe deficiency in the shoot [15]. These results indicate that IAA could intervene in both the shoot to root activating signal promoted by the presence of Fe deficiency in shoot, and the key factor that triggers the molecular events associated with the regulation of physiological Fe-stress responses in the root. Other results obtained in diverse plant species

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also support this potential role of auxin as a candidate for the shoot to root activating signal [16]. Auxin may work through other phytoregulators that would act as second messengers [16,17]. The important role of ethylene in the activation-regulation of Fe-stress root responses has been demonstrated in diverse plant species [18]. Ethylene action seemed to be coupled with a concomitant regulatory action of nitric oxide (NO) [19]. This action of NO seems to be involved in auxin-dependent activation of Fe(III)-chelate reductase in arabidopsis [15]. However, the activating role of ethylene and NO was repressed under conditions of Fe sufficiency [19]. This repression may be mediated by the presence of Fe in the phloem [19,20]. These results suggest that both ethylene and NO might have relevant roles in the signaling and regulation of physiological Fe-stress responses into the root area [21]. However, the activating action of exogenous IAA is not blocked under conditions of Fe sufficiency, suggesting that IAA may also act through an ethylene-NO independent-alternative pathway [15]. Jasmonic acid and some cytokinins could be involved in repressing the process [22,23]. The application of exogenous cytokinins blocked the expression of AtFRO2 and AtIRT1 [22]. Methyl-jasmonate applied to wild type and certain mutants of arabidopsis had a similar effect [23]. However, either the action of jasmonate or that of cytokinins was independent of FIT expression. This fact makes it complicated to interpret their roles in the whole regulatory network of some Fe deficiency plant responses. In any case, the role of both cytokinins and jasmonate may be complementary and secondary to that of shoot to root repressing signal. The physiological event associated with Fe deficiency that triggers the synthesis and shoot to root translocation of the activating signal could be related to a physiological damage caused by Fe deficiency and/or the concentration of a specific pool or pools of Fe in the shoot. The physiological disorders caused by Fe deficiency in the plant are often common to the deficiency of other nutrients (for instance, Mg in the case of chlorophyll synthesis and photosynthetic activity). Thus, it is more probable that this process is directly related to the concentration of one, or several, pools of Fe (mainly those related to metabolic available Fe) in plant shoot (principally leaves) (Fe-specific signal). This fact is indirectly supported by the presence in some plant species of the so called Fe chlorosis paradox consisting of the coexistence of high concentrations of Fe in leaves and Fe chlorosis [24]. This contradiction often disappears when the concentration in leaf of easily soluble Fe (the fraction of Fe in leaves that is extracted by 0.5 M HCl) is explicitly considered instead of total Fe leaf concentration [25]. Easily soluble Fe is assumed to be directly related to the pool of Fe that is available for biochemical processes [25]. In this sense this concept is analog to that related to cellular inorganic phosphate content (Pi) [26]. This concept involves those pools of phosphorus (P) that are not incorporated to structural or organic metabolites within the plant. It is analyzed by submerging shoot or root tissues in 1 ml of 1% glacial acetate and freeze-thawed eight times [26]. A number of studies have proven that Pi and total P are complementary parameters, with Pi being more useful for studies on P root uptake, signaling and metabolism [26]. In any case, there is no experimental evidence that permits elucidating the real nature of the factor sensing the deficiency of Fe available for metabolic processes in plant shoot. The main question is whether the activation of Fe-stress root responses is directly linked to a decrease in Fe availability in root that is associated with both a decrease in Fe in the xylem and Fe available for biochemical processes in the leaf, or this step is mediated and regulated by the activation of a specific system focused on the optimization of Fe metabolic use efficiency.

Table 1 Shoot and root growth indexes and time-course activation of Fe(III)-chelate reductase activity for two cucumber varieties cultivated under Fe starvation (the table has been made from data published in Ref. [4]). Parameters

Cucumber variety

a

Shoot growth index Root growth indexb Day of activation of Fe(III)-chelate reductasec

Ashley

Anico

4.60 3 19

1.10 1.07 12

a (Shoot dry weight at the end of the experiment)/(Shoot dry weight at the beginning of the experiment) (from Ref. [4]). b (Root dry weight at the end of the experiment)/(Root dry weight at the beginning of the experiment) (from Ref. [4]). c First day of significant activation of Fe(III)-chelate reductase activity after the onset of the experiment (from Ref. [4]).

4. Experimental evidence supporting the existence of a system of optimized metabolic use of Fe in the shoot, which co-regulates the activation of (Strategy I) physiological Fe-stress root responses In general, “Fe-efficient plant variety” concept has been linked to the plant ability to take up Fe from the rhizosphere through an effective and intense activation of Fe-stress root responses [27]. Some studies employing diverse plant species did not find clear relationships between plant Fe-efficiency and the ability of the plant to activate Fe-stress root responses [28]. A Fe efficient plant variety can be also defined as a variety able to grow under conditions of limitation in available Fe [28]. In consequence plant Fe efficiency seems to involve two complementary abilities: (i) the ability to grow under Fe limiting conditions; and (ii) the ability to activate specific root responses in order to facilitate the uptake of Fe from the soil. The ability to grow in the absence of Fe in the nutrient solution (or in the presence of very low Fe concentrations) involves the optimization of the metabolic use of the Fe present within the plant (root and shoot). Several studies carried out in our laboratory dealt with Fe nutrition in several cucumber plant varieties subjected to Fe-deficient conditions [4]. The main aim of these studies was to select two different varieties with diverse Fe efficiency (high and low) to explore the hormonal balance in these plants under Fe shortage [4]. The results obtained showed that these cucumber varieties have both the ability to activate physiological and morphological Fe-stress root responses and the ability to grow, under Fe limiting conditions. However, cucumber varieties had a diverse ability to grow under Fe-shortage, although Fe-leaf concentration was not different among them at the beginning of the experiment [4]. Unexpectedly, the activation of some physiological Fe-stress root responses seemed to be directly affected by the ability of each variety to both grow and take up nutrients under these conditions [4]. Thus, the most inefficient variety for growing under Fe starvation expressed a rapid and intense activation of Fe(III)-chelate reductase [4] (Table 1). Conversely, the most efficient variety to grow under Fe starvation seemed to delay the activation of Fe(III)-chelate reductase [4] (Table 1). The Fe concentration (both total and easily soluble fraction) in Fe-inefficient plants was even higher than in Fe-efficient plants, probably due to the production of less dry matter and nutrient accumulation-concentration. These results suggest that the use of Fe in physiological processes in Fe-efficient plants is much better than in Fe-inefficient plants. These results, taken together, also support that the two processes involved in plant Fe efficiency ability are complementary and, probably, co-regulated. In this sense, it is especially interesting the work of Baxter et al. [29]. These authors explored the usefulness of the ionomic footprint of arabidopsis varieties growing in the presence of diverse concentrations of available Fe in the nutrient solution.

J.M. García-Mina et al. / Plant Science 198 (2013) 39–45 Table 2 Influence of phenolics removal on the intensity of the time-course activation of Fe(III)-chelate reductase in roots of red clover plants subjected to Fe-deficiency (the table has been made from data published in Ref. [30]; values of Fe(III)-chelate reductase are expressed in arbitrary-comparative units) (* indicates significant differences for p < 0.05 t test). Treatments

Without Fe Without Fe plus phenolics removal

Days 4

8

12

16

2.6 3.6*

1.9 3.4*

1 2.2*

0.5 2*

Results showed that plants receiving different Fe concentrations (1 up to 10 ␮M) tended to maintain the same concentration of Fe in the shoot. This fact indicates that in the presence of available Fe plants both optimize Fe concentration in shoot and activate Fe-root uptake (the activation of specific root responses to Fe deficiency will depend on the concentration of available Fe in the nutrient solution) [29]. This study also showed the usefulness of considering the whole ionome and nutrient concentration relationships to detect plant varieties with modifications in those mechanisms involved in Fe and P homeostasis [29]. On the other hand, there is experimental evidence supporting that the optimization of Fe-metabolic use in the shoot may include, or be coupled with, the remobilization of inactive or stored Fe pools within the plant. The role of the biosynthesis and root accumulation of phenolics on the remobilization of apoplastic Fe in the control of some physiological Fe-stress root responses in red clover was studied [30]. When phenolic compounds were removed from the root-nutrient solution, plants activated some physiological Festress root responses faster than in the presence of phenolics. The action of phenolics, contributing to the remobilization of inactive Fe pools mainly in the apoplast and cell-wall in the root, delayed the activation in the root of Fe(III)-chelate reductase and also affected rhizosphere acidification [30]. The remobilization of unavailable Fe within the plant maybe involved in the intermediate regulatory system focused on the optimization of Fe-metabolic use in plant shoot. This process would involve the biosynthesis of specific compounds that solubilize precipitated or immobilized Fe present within the plant [2,30]. Lemon trees subjected to Fe chlorosis have a distribution of these types of compounds (mainly phenolics) within the plant that included many different organs, also affecting fruit composition and quality regarding antioxidant activity [31]. This study also showed that the presence of specific phenolics in diverse plant organs is an indicator or signal of Fe chlorosis intensity [31]. The open question is if the system involved in the optimization of the metabolic use of Fe in the shoot is expressed before, after, – or simultaneously co-regulated – of the activation of physiological Fe-stress root responses. There is no enough experimental evidence to elucidate this point. However, the above-mentioned studies involving cucumber [4], and red clover [30], allow us to propose a provisional hypothesis. In the study on the Fe efficiency of diverse cucumber varieties, the activation of Fe(III)-chelate reductase was expressed before and faster in those varieties that presented a poor ability to grow under Fe starvation compared with the most efficient variety to grow without Fe [4] (Table 1). The study of the role of phenolics and apoplastic Fe remobilization in the expression of root responses to Fe starvation in red clover showed that when phenolics were removed from the root and nutrient solution, plants activated Fe(III) chelate reductase with more intensity than those plants without phenolics removal [30] (Table 2). These results, taken together, suggest that, at least at the beginning of Fe deficiency conditions, the system focused on the

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optimization of Fe-metabolic use in the shoot is probably activated before the activation of the main physiological Fe-stress root responses. Likewise, plant efficiency to optimize the metabolic use of Fe in the shoot may regulate physiological Fe-stress root responses by affecting the intensity of Fe-deficiency physiological consequences in the shoot and, consequently, the synthesis and shoot to root translocation of the signal responsible for the activation of some physiological root responses to Fe starvation (Fig. 1C). This model is compatible with the alternate activation of both coregulated systems during plant cycle in strategy I-crops cultivated in Fe-deficient soils.

5. Potential consequences of the existence of a system to optimize the metabolic use of Fe in the shoot in Fe-fertilization of Strategy-I crops Complementary information that might illustrate the potential practical consequences of the coordinated model hypothesis is the behavior of plants cultivated under Fe deficient conditions, which received potentially available Fe in water-insoluble forms. The remediation of Fe chlorosis in lemon trees cultivated in calcareous soils was studied using a hetero-molecular chelate that contained potentially available Fe for plants, but that is retained in soil matrix thus presenting very low Fe concentration in soil solution [32]. The expected advantage of this chelate is that due to soil fixation all processes linked to leaching are diminished [32]. The authors compared in this work the effects of the hetero-molecular Fe chelate on lemon trees Fe nutrition with those of a highly available Fe chelate (Fe-EDDHA). The hypothesis behind the use of this type of chelate (hetero-molecular Fe chelate) was based on the coordinated model. It was assumed that lemon trees have to activate Fe-stress root responses to take up chelated Fe from these hetero-molecular chelates, because the concentration of Fe in soil solution is very low. Lemon trees receiving Fe-EDDHA do not need to activate Fe-stress root responses. According to coordinated model citrus plants receiving Fe from the hetero-molecular chelate will activate the regulatory system involved in the optimization of Feuse in the shoot, saving energy. In this way, these plants optimize the metabolic use of the Fe present within the plant (including remobilization) before activating Fe-stress root responses. This fact would imply a continuous equilibrium or cross-talk between the system to optimize the use of Fe in the shoot in biochemical processes and Fe-stress root responses. This fact will be reflected in the ratio between the fruit yield and easily soluble Fe concentration in leaves for trees treated with the hetero-molecular chelate and control trees receiving easily available Fe (Fe-EDDHA). If this hypothesis is true, this ratio will be higher for the hetero-molecular Fe chelate treatment for obtaining the same yield [32]. The results obtained confirmed this hypothesis. Lemon trees receiving Fe as the natural hetero-molecular Fe chelate produced the same yield and fruit quality as those trees receiving a highly water-soluble and stable Fe chelate, but with a lower concentration of easily soluble Fe in young leaves [32] (Table 3). This means that the system for the optimization of the metabolic use of Fe in the shoot affects the concentration of Fe in leaf that is necessary to produce final yield. This optimization process is compatible with the fact that the activity of physiological Fe-stress root responses is probably cyclic due to the regulatory crosstalk between Fe-efficient use optimization in the shoot and Fe-stress root responses during plant growth [33]. These results also showed that this process does not negatively affect yield but, even, it may contribute to optimize Fe plant nutrition. These results were confirmed in field studies with orange and peach trees (Table 3). There is no experimental evidence that may permit us to propose specific candidates for the signal that activates the

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Table 3 Easily soluble-Fe concentration in young leaves, final yield and plant metabolic-use efficiency of Fe for three field experiments: lemon trees [32], orange trees and peach trees (unpublished results) (Fe-EDDHA corresponds to the treatment with the Fe-chelate Fe-EDDHA; HMC corresponds to the treatment with the hetero-molecular Fe chelate). Young leaf active Fe (mg kg−1 )

Field experiments treatments Lemon Trees Fe-EDDHA HMC Orange Trees Fe-EDDHA HMC Peach Trees Fe-EDDHA HMC

Yield (kg per Tree)

202.80 218.60

15.90 14.70

Metabolic-use efficiency. Yield/active Fe (percentage considering Fe EDDHA as 100) 12.70 (100) 14.90 (117)

34.37 13.51

94 91

2.73 (100) 6.74 (247)

74.07 39.52

73 68

0.98 (100) 1.72 (176)

Easily soluble Fe concentration is obtained by extraction of Fe with cold 0.5 M HCl [32].

Table 4 Shoot dry weight, phosphorus (P) concentration in the shoot and plant metabolic-use efficiency of P for diverse plant species cultivated in the presence of monopotassium phosphate (KP) or a phosphate-Fe-humic complex (PFeHA), in the nutrient solution (data from Ref. [35]). Plant treatments Wheat KP PFeHA Chickpea KP PFeHA

P concentration in shoot (P) (g kg−1 )

Shoot dry weight (SDW) (mg per plant)

Metabolic-use efficiency. SDW/P (percentage considering KP as 100)

8.70 4.12

120 119

13.80 (100) 28.90 (209)

5.16 2.94

1733 1963

335 (100) 668 (199)

optimization of Fe-metabolic use in the shoot. However, the signal 6. Conclusions and perspectives might be directly associated with the impairment of one specific, or several, physiological processes in the shoot, which in The results discussed here are compatible with a hypothesis turn, may be coupled with a parameter related to a Fe fraction proposing the existence of a specific regulatory network-system related to Fe available to be used in biochemical processes in the that optimizes Fe-use in the shoot for biochemical-metabolic proleaf. This Fe pool could partially be evaluated by the easily-soluble cesses, which would be expressed probably before the activation Fe fraction. This process could be related to some of the reacof physiological Fe-stress root responses (Fig. 1C). This system tive species produced in the Fenton reaction where Fe plays an would regulate (or co-regulate) some Fe-stress root responses and important role. May be, the lack of some of these reactive species could play a key role in both Fe plant distribution and trafficking, could trigger the activation of the intermediate regulatory system mainly under Fe limiting conditions, and allow remediation of that optimizes Fe-use in the shoot and Fe-remobilization within Fe-chlorosis. In this sense, studies oriented to establish an anathe plant. Further studies are needed in order to elucidate this lytical method for the determination of the Fe pool in shoot and question. root that is easily available to be used in biochemical processes are Results of experiments dealing with P nutrition, using watervery relevant and necessary. An analytical approach could be Fe insoluble P fertilizers, were similar to those related to Fe extraction with cold diluted HCl and named here easily soluble Fe [34,35] (Table 4). These results suggest that a specific coor[25]. However, another possibility is to use the method employed dinated type model could also be involved in P acquisition to determine cellular inorganic phosphate that has proved to by plants cultivated under conditions of P limited availability be very efficient in studies on the regulation of P root uptake, [35]. transport and assimilation [26]. Finally, experimental studies specifically designed to explore The practical definition of plant-related nutrient use efficiency and fertilizer-related efficiency, are also affected by the existence of the existence and features of the system that optimizes Fean intermediate regulatory system that optimizes nutrient-use in metabolic use in the shoot, and its role and integration in the the shoot. Two factors should be considered to define these paramregulation of Fe plant use and root uptake under conditions of Fe eters. It is important to focus attention on the overall nutrient-use limitation, are needed in order to elucidate the real importance of plant efficiency, which can be defined as the ratio between yield this intermediate regulatory system, its regulation and potential and the concentration of nutrient in the shoot that is available implications in Fe-remediation in field Strategy I-crops. for biochemical processes. It is important to take into account the residual fraction of potentially plant-available nutrient that rest Acknowledgements in soil after harvesting, with respect to the fertilizer added to the soil. This work has been supported by Roullier Group, CDTI and GovTherefore, we could define a factor to evaluate the whole ernment of Navarra. We would like to thank all comments and fertilizer-plant nutrient use efficiency (FP-NUE) as equaling   the: Concentration of nutrient that is available to be Yield × used in biochemical processes in the leaves or shoot

 Residual

plant available nutrient after harvesting total nutrient added to the crop



× 100. This parameter demands the development of adequate analytical methods to measure both the fraction of nutrient in the shoot available for biochemical processes, and the residual fraction of plant available nutrient in soil after crop harvesting.

suggestions done by the reviewers, because they have contributed to significantly improve the quality of the manuscript. Finally we would like to thank D. Rhymes for helping us in English redaction.

J.M. García-Mina et al. / Plant Science 198 (2013) 39–45

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