It’s time to make changes: modulation of root system architecture by nutrient signals

Abstract

Root growth and development are of outstanding importance for the plant’s ability to acquire water and nutrients from different soil horizons. To cope with fluctuating nutrient availabilities, plants integrate systemic signals pertaining to their nutritional status into developmental pathways that regulate the spatial arrangement of roots. Changes in the plant nutritional status and external nutrient supply modulate root system architecture (RSA) over time and determine the degree of root plasticity which is based on variations in the number, extension, placement, and growth direction of individual components of the root system. Roots also sense the local availability of some nutrients, thereby leading to nutrient-specific modifications in RSA, that result from the integration of systemic and local signals into the root developmental programme at specific steps. An in silico analysis of nutrient-responsive genes involved in root development showed that the majority of these specifically responded to the deficiency of individual nutrients while a minority responded to more than one nutrient deficiency. Such an analysis provides an interesting starting point for the identification of the molecular players underlying the sensing and transduction of the nutrient signals that mediate changes in the development and architecture of root systems.

Lateral root, local signalling, nutrient deficiency, primary root, root development, root traits, systemic signalling.

Introduction

Roots are the major determinants for a balanced nutrition of plants, since they explore underground in the search for water and nutrients. Thereby, roots have a massive impact on the growth and development of plants as well as on the plant’s adaptation to environmental constraints. Despite the vital importance of roots, breeding efforts for the improvement of root systems for the sake of stress tolerance and, ultimately, yield gain and stability have only recently started compared with the breeding programmes targeting above-ground traits. This relatively late interest in roots mainly goes back to the difficulty in accessing intact root systems for analysis, particularly under field conditions (de Dorlodot et al., 2007; Lynch, 2007).

The root system architecture (RSA) of a plant refers ultimately to the three-dimensional placement of roots in the soil and comprises aspects of root morphology, topology, and distribution (Lynch, 1995). At any given time, the characteristic RSA of a plant results from the combination of its genetic background and the prevailing environmental conditions experienced by that particular plant (Malamy, 2005). During the lifetime of a plant the RSA undergoes alterations in the elongation, branching, and spacing of roots of different orders, ultimately modifying the overall shape of a root system over time. Thus, root systems can exhibit a large degree of developmental plasticity which is a consequence of the perception and integration of environmental information into the root developmental programme (Novoplansky, 2002). Root plasticity allows plants to modulate the three-dimensional arrangement of the root system in order to optimize growth in a heterogeneous and constantly changing environment.

This review focuses on the latest advances made in the identification of nutrient-sensitive steps in the modulation of root architectural traits. Using an in silico approach, a set of genes is also described that appear as candidates for the integration of nutrient signals into the root developmental programme of Arabidopsis thaliana.

Morphological and topological components of the RSA and their underlying biological processes

Primary root length

The length of a root is determined by the combination of three major cell biological processes: the rate of cell division, the rate of cell differentiation, and the extent of expansion and elongation of cells (Scheres et al., 2002). In dicotyledonous plants, the development of root systems starts with an embryonic primary root meristem. At this stage, the positioning and specification of the stem cell niche (SCN), formed by the quiescent centre (QC) and the surrounding stem cells (initial cells), are established (Scheres et al., 1994). This developmental process is followed by post-embryonic events that produce a so-called transit amplifying cell population in the proximal meristem. Since the root apical meristem (RAM) is the ultimate reservoir of all cells that will constitute a primary root throughout its lifetime, perturbations in the spatial arrangement and functional maintenance of the RAM will ultimately affect primary root growth.

Mitotic activity in the RAM determines the extent and direction of primary root growth. One important observation is that cell division decreases as cells progress further away from the SCN (Petricka et al., 2012). Eventually, cells stop dividing and undergo differentiation and elongation. The position where cell elongation starts in relation to the SCN position determines the size of the meristem which, in turn, is directly related to the rate of root growth (Beemster and Baskin, 1998). The establishment and maintenance of the RAM is orchestrated by pathways involving hormones and developmental genes. Auxin is pivotal to the regulation of root development. The polar and cell type-specific distribution of PINFORMED (PIN) auxin efflux carriers generates an auxin concentration gradient along the root axis, with a maximum at the root apex (Sabatini et al., 1999). Two AP2-domain transcription factors, PLETHORA (PLT) 1 and 2, show an auxin dose-dependent accumulation and have their highest expression overlapping with the auxin maximum in the RAM (Galinha et al., 2007). Importantly, the highest PLT levels specify the positioning of the SCN, whereas the lowest PLT abundance determines where cells start to differentiate (Aida et al., 2004; Galinha et al., 2007). Parallel to PLT1 and PLT2, the transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) are also involved in the maintenance of the SCN (Helariutta et al., 2000; Sabatini et al., 2003), as well as in the specification of the endodermis (Cui et al., 2007). In addition to their role in RAM patterning, SHR and SCR have also been found to regulate the expression of the cell cycle regulator CYCLIN D6;1 (CYCD6;1) directly, thus interconnecting patterning and growth in the root tip (Sozzani et al., 2010).

The interplay between auxin and cytokinin determines whether meristematic cells divide or undergo differentiation (Dello Ioio et al., 2007; Ruzicka et al., 2009). This developmental decision involves the down-regulation of PIN1, PIN3, and PIN7 by the repressor of auxin signalling SHORT HYPOCOTYL2 (SHY2) (Dello Ioio et al., 2008). Because cytokinins promote SHY2 expression and auxin directs SHY2 for degradation (Dello Ioio et al., 2008), the concerted action of the two hormones maintains a balance between cell division and differentiation. Once cells lose their proliferative capacity, they undergo expansion and elongation. This process is highly dependent on the development of a central vacuole (Schumacher et al., 1999) and on cell wall modifications (Cosgrove, 2000; Fagard et al., 2000).

Lateral root (LR) density

Besides primary root length, the number and length of LRs represent the other dominant feature of RSA. Although fully developed LRs are structurally very similar to primary roots, the formation of a LR is a completely post-embryonic event (Peret et al., 2009; De Smet, 2012; Petricka et al., 2012). The formation of a new LR involves a series of tightly co-ordinated events which start with the pre-initiation and are followed by the initiation, development, and emergence of the LR primordium (Peret et al., 2009). The initial step of LR development takes place in the basal meristem of the parental root, where xylem pole pericycle cells are ‘primed’, i.e. acquire the capacity to form a LR upon activation (De Smet et al., 2007). It has recently been shown that the priming of pericycle cells coincides with the oscillatory expression of a large set of genes within the so-called oscillation zone, a region in the parental root which comprises the basal meristem and the elongation zone (Moreno-Risueno et al., 2010). Interestingly, auxin also oscillates in the basal meristem (De Smet et al., 2007; Moreno-Risueno et al., 2010). However, this hormone alone is not determining the position of LR pre-branch sites (Moreno-Risueno et al., 2010), suggesting that some of the oscillatory genes may be key regulators of this process. Until now it is not clear whether the number of LR pre-branch sites can also be affected by environmental cues or whether such cues only alter root branching by modulating the subsequent steps of LR formation.

Auxin is required for the activation of LR pre-branch sites that then commence with LR formation (Casimiro et al., 2001; Dubrovsky et al., 2008). The cell type-specific distribution and levels of auxin required to promote LR initiation are sustained by the auxin carriers AUX1 and PINs (Laskowski et al., 2008). Auxin is then perceived and activates a set of genes that regulate the subsequent steps of LR formation. Firstly, auxin perception triggers the degradation of IAA14/SLR, thereby de-repressing the expression of the transcription factors ARF7 and ARF19 (Fukaki et al., 2005). Their corresponding gene products activate the expression of another set of genes, including the transcription factors LBD16/ASL18 and LBD29/ASL16 (Okushima et al., 2007) which, in turn, are required for the asymmetrical cell division that gives rise to a shorter and a longer daughter cell (Goh et al., 2012). The shorter cells are highly sensitive to auxin and express ACR4, a leucine-rich repeat receptor-like kinase that represses cell division in the adjacent pericycle cells (De Smet et al., 2008). Then, a precise sequence of cell divisions takes place to form the new LR primordium, a process that requires the auxin-regulated transcription factor PUCHI (Hirota et al., 2007).

Again, auxin is involved in reprogramming cells adjacent to the new LR primordium to facilitate its emergence as it breaks through three overlaying cell layers (Peret et al., 2009). To aid in this process, auxin originating from the LR primordium activates cell wall-remodelling enzymes that loosen the adjacent cells (Swarup et al., 2008). In order to maintain the overall integrity of the parental root, the auxin-induced cell wall remodelling is tightly restricted to the cells directly overlaying the LR primordium. This is achieved by the confined expression of the auxin carrier LAX3, which then accumulates auxin specifically in these cells (Swarup et al., 2008). In addition to auxin, other hormones such as cytokinin, ethylene, and ABA play a role during the different steps of LR formation by positively or negatively interacting with auxin (reviewed by Fukaki and Tasaka, 2009).

Lateral root length

The last step of LR formation is the emergence of LRs and the activation of their meristems. The activation of LR meristems may relate to the ability of LRs to increase their own auxin synthesis allowing them to escape the apical dominance of the primary root. This assumption is in line with the fact that, although the post-emergence development of LRs is lost in alf3 mutants, it can be rescued by exogenously supplying auxin to these plants (Celenza et al., 1995). However, sustained development and elongation of LRs still depends on auxin originated from the shoot and/or primary root, as suggested by slower elongation of LRs upon mutation of the rootward auxin transporter MDR1 (Spalding et al., 2007). At later developmental stages LRs resemble primary roots and it is thought that most of the developmental processes that regulate primary root growth are also involved in LR growth. However, it is noteworthy that LRs exhibit some unique features, such as an altered gravitropic response (Mullen and Hangarter, 2003; Rosquete et al., 2013). The nature of the molecular players underlying this and other possible distinctions between the primary and LRs remains to be resolved.

The modulation of root development by nutrients

To sustain their growth and development, plants must take up 14 mineral elements from the soil. This necessity is complicated by the fact that the availability of most nutrients within the soil profile fluctuates in a spatial and temporal manner (Burns, 1980; White et al., 1987). Thus, in order to sustain optimal growth and development, plants need constantly to monitor and respond to these fluctuations. Once detected, nutrient-derived signals trigger physiological responses in order to optimize nutrient mobilization, uptake, storage, and allocation. Molecular mechanisms underlying nutrient sensing and signalling have recently been reviewed (Krouk et al., 2010a; Alvarez et al., 2012; Schachtman, 2012). Regarding the effect of nutrients on morphological responses, there is growing evidence that the information on the nutritional status of the plant, as well as the spatial availability of nutrients in the surrounding environment, evoke changes in the overall RSA. Such targeted rearrangements of the root system within the soil help plant roots to optimize a spatially defined exploration of the most favourable sites.

Nutrient deficiencies: when systemic signals control RSA

Nutrient availability can have a profound impact on RSA by altering the number, length, angle, and diameter of roots (reviewed by Forde and Lorenzo, 2001; Lopez-Bucio et al., 2003; Malamy, 2005; Osmont et al., 2007). As an example, the root system of Arabidopsis thaliana plants grown under limited phosphorous (P) is commonly shallower, as P deficiency inhibits primary root elongation and stimulates LR formation (Williamson et al., 2001; Lopez-Bucio et al., 2002; Sanchez-Calderon et al., 2005; Gruber et al., 2013). Thereby, P deficiency up-regulates the expression of the auxin receptor TIR1 and thus increases the sensitivity of pericycle cells towards auxin (Perez-Torres et al., 2008). Finally, this leads to the activation of ARF transcription factors that promote LR initiation and emergence. The inhibition of primary root length under low P results initially from decreased cell elongation and, subsequently, from slower cell division rates in the RAM (Sanchez-Calderon et al., 2005). In the same study, it was also found that the reduced mitotic activity observed in P-deficient primary root tips was associated with alterations in the QC. In fact, under low P availability, the P5-type ATPase PDR2 maintains nuclear SCR levels in the RAM, thus keeping a balance between cell division and differentiation (Ticconi et al., 2009). Under P-deficient conditions in the field, plants with a shallower root system may be more adapted to explore the P-enriched topsoil (Lynch and Brown, 2001; Rubio et al., 2003; Zhu et al., 2005).

In contrast to P limitation, low nitrogen (N) availability stimulated the elongation of primary and LRs in particular, whereas LR density remained largely unaffected (Linkohr et al., 2002; Lopez-Bucio et al., 2003; Gruber et al., 2013). Such RSA modifications are thought to improve the plant’s ability to forage the soil more efficiently in the search for sparingly available nutrients, or to extract N before it leaches out of the rooting zone in what is now a part of the ‘steep, cheap, and deep’ root ideotype proposed for maize (Wiesler and Horst, 1994; Lynch, 2013).

So far, the characterization of the effects of most nutrients on roots has mainly been restricted to measurements of root biomass and total root length (Hermans and Verbruggen, 2005; Hermans et al., 2006; Richard-Molard et al., 2008; Jung et al., 2009; Cailliatte et al., 2010). In an attempt to systematically characterize the modifications that the RSA undergoes when the availability of nutrients is decreased, an approach has recently been undertaken to determine the plasticity of the root system under different nutrient deficiencies (Gruber et al., 2013). It was observed that RSA changes to different degrees depending on the type and quantity of the nutrient being withdrawn. Calcium deficiency caused the largest change in individual RSA components, despite the absence of any change in the total root length. On the other hand, sulphur (S) deficiency produced small changes in RSA, even if the shoot was severely affected by the deficiency, an effect also reported in crop species (Kutschera et al., 2009). The deficiency of single nutrients had distinct effects on the different components of the RSA (Fig. 1). Noteworthy, the level of deficiency dictates the degree of the RSA response (Gruber et al., 2013). This suggests that active foraging strategies can only be sustained if a certain ‘critical’ amount of the nutrient in question is available to plants. One open question is to what extent RSA modifications observed in nutrient-deficient plants result from the lack of a nutrient’s biochemical and physiological function in metabolism or from signalling events that alter RSA in a way to mitigate the deficiency.

Fig. 1.

Schematic representation of the main effects of nutrients on root system architecture in relation to whether they are homogeneously or heterogeneously available for plants. For most nutrients, the extent of these effects depends on the nutrient concentration supplied to plants (Gruber et al., 2013). The limitation of one nutrient can have opposing effects on different RSA traits. In addition, it has been shown for some nutrients that their local availability can affect the elongation or branching of roots growing into the nutrient-enriched patch. The darker patches on the right side of the figure indicate an increased availability of a particular nutrient. : nitrate; : ammonium; B: boron; Mg: magnesium; Mn: manganese.

Interestingly, although primary root length was reduced under low calcium (Ca) supply, as also commonly reported for P deficiency (Williamson et al., 2001; Lopez-Bucio et al., 2002; Sanchez-Calderon et al., 2005), the elongation of the first order lateral roots was maintained in Ca-deficient plants (Gruber et al., 2013). This indicates that developmental changes of different orders of roots are primarily uncoupled, an effect that was indeed observed in a number of nutrient deficiencies. With regard to the deficiency of a particular nutrient, it may be assumed that the underlying developmental processes, or at least the nutrient-dependent regulation thereof, are subject to different sensitivities depending on the order of a root. It has recently been shown that potassium (K) starvation represses LR elongation independently of the genetic background, whereas the effect of K deficiency on primary root length ranged from slightly to severely repressed, depending upon the accession line (Kellermeier et al., 2013). These observations indicate that genotypic differences influence the extent of RSA responses to the plant nutritional status, probably reflecting differences in both sensitivity and responsiveness.

Heterogeneous nutrient availability: when local signals act on top of systemic signals to control RSA

The ability of plant roots to respond to a localized availability of nutrients was demonstrated in early studies where nutrients were heterogeneously supplied in order to be accessible to only a part of the root system (Drew, 1975; Drew and Saker, 1975; Granato et al., 1989; Burns, 1991; Robinson, 1994). Similar experiments were also carried out in vitro with Arabidopsis thaliana (Zhang and Forde, 1998; Linkohr et al., 2002; Remans et al., 2006; Giehl et al., 2012a; Lima et al., 2010). In the case of nitrate, the local availability stimulated the elongation of those LRs that were growing into the nitrate-containing agar medium (Zhang and Forde, 1998; Linkohr et al., 2002). This root architectural modification was not only related to a stimulation of LR elongation within the nitrate-containing compartment, but also associated with a repression of lateral roots growing outside the nitrate patch. Rather than reflecting a nutritional effect, the response of roots to nitrate appears to be primarily the outcome of a signalling cascade activated by nitrate sensing (Zhang and Forde, 1998; Zhang et al., 1999).

Besides nitrate, it has been shown that a local availability of another inorganic N form, ammonium, also elicits modifications in LR development (Lima et al., 2010). In contrast to nitrate which mainly stimulates LR elongation (Zhang and Forde, 1998; Zhang et al., 1999; Remans et al., 2006), the supply of ammonium to a portion of the roots induced mainly LR initiation (Lima et al., 2010). Thus, these two inorganic N forms seem to have complementary effects on RSA (Fig. 2). As for nitrate, the effect of ammonium on root branching is probably associated with a signalling rather than a nutritional effect (Lima et al., 2010). In addition, the effect of ammonium on LR branching was strongly promoted by the ammonium transporter AMT1;3, placing this transporter upstream on the signalling cascade that regulates RSA in response to ammonium.

Fig. 2.

The effect of nutrients on the expression of genes involved in root development. A Venn diagram depicting the overlap of RDR genes (at least 2-fold up- or down-) regulated by the indicated nutrient deficiencies. The numbers within one circle or more than one circle indicate genes specifically regulated by one nutrient deficiency or shared by more than one nutrient, respectively. All nutrient-regulated RDR genes are indicated and those written in blue were specifically regulated by the indicated condition. The RSA component affected by each gene is indicated, where MR=mature root development (patterning, cell division, differentiation, and/or elongation); LRF=LR formation (LR initiation and/or LR emergence); other=general effect on RSA. Transcriptional data were obtained from Genevestigator (http://www.genevestigator.ethz.ch). Venn diagrams were prepared with Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html).

More recently, it has been shown that a spatial restriction of the micronutrient iron (Fe) also evokes RSA modifications in plants (Giehl et al., 2012a, b). Similarly to nitrate and P, localized Fe supply targeted LR elongation rather than LR density (Fig. 1). Interestingly, auxin accumulation was favoured in those LRs that had access to Fe, suggesting an effect associated with an Fe-dependent up-regulation of the auxin carrier AUX1 in LR apices (Giehl et al., 2012a).

Whereas nitrate is highly mobile in soils, the interaction of other nutrients, such as ammonium, phosphate, and Fe, with soil components significantly reduce their mobility (Tinker and Nye, 2000). Differences in soil mobility could have directly driven the evolution of distinct RSA responses in plants. However, it is noteworthy that an uneven distribution of relatively immobile nutrients like Fe and ammonium induce different changes in RSA, namely LR elongation and initiation, respectively (Lima et al., 2010; Giehl et al., 2012a). Similar discrepancies also hold true for Fe and nitrate, two nutrients with contrasting mobilities in soil (Tinker and Nye, 2000), but which, nevertheless, target mainly LR elongation (Zhang and Forde, 1998; Giehl et al., 2012a). Thus, perhaps the characteristic mobility of a nutrient in soils was not the driving factor for the evolution of RSA responses to uneven nutrient availabilities. Instead, there is growing evidence in grasses to suggest that the exploitation of patchy nutrient reserves provides a competitive advantage over other individuals, particularly those from other plant species (Hodge et al., 1999; Robinson et al., 1999). Plants typically devote more effort into exploiting higher quality patches of nutrients (Jackson and Caldwell, 1989; McNickle and Cahill, 2009) and can do so very quickly, as evident from a study with grasses that showed a 4-fold increase in the root growth rate just 1 d after the establishment of a patchy nutrient supply (Jackson and Caldwell, 1989). Therefore, the ability to exploit a patch of immobile nutrients quickly or to extract a mobile nutrient from a patch before it diffuses and would become accessible to competitors may well have provided the selective pressure needed to develop such root traits in plants.

How nutrient signals are integrated into root developmental processes

Despite growing evidence indicating that the availability of nutrients can modulate root growth and architecture in plants, it is still poorly understood how this modulation takes place. Perhaps the most plausible mechanistic view is that, once perceived, nutrient signals eventually affect a set of genes/proteins in the root developmental programme in order to modify specific root developmental processes. This view is in agreement with RSA modifications observed under heterogeneous Fe supply: While Fe deficiency represses the elongation of LRs, local Fe supply restores elongation only in those LRs that have access to Fe and induce AUX1 to promote auxin accumulation (Giehl et al., 2012a). This process is inhibited as soon as the remaining root system or the shoot is resupplied with Fe, indicating that a systemic signal reflecting a deficient Fe nutritional status is prerequisite for the local action of Fe. As further suggested by the use of auxin transport inhibitors, it is primarily the rootward auxin flow originating from the shoot apical meristem that is deviated by the local up-regulation of AUX1 into Fe-supplied LRs to promote their elongation (Giehl et al., 2012a). Thus, AUX1 represents some sort of check-point where systemic and local nutritional signals are integrated into the root developmental programme.

In an attempt to identify other genes involved in RSA responses to nutrient availabilities, a search was made for nutrient-regulated developmental genes expressed in roots. For this purpose, use was made of the transcription information that is publicly available in Genevestigator (Zimmermann et al., 2004). Among the experiments present within the ‘perturbations’ category, transcriptome analyses from 77 nutrient-related conditions are currently available (http://www.genevestigator.ethz.ch; access June, 2013). From these, 40 relate to conditions in which plants were grown under the deficiency of five nutrients, –N (disregarding split-root experiments), –P, –Fe, –S or –K (see Supplementary Table S1 at JXB online). Using these transcriptome data, the transcriptional changes of 98 root development-related (RDR) genes were examined, for which genetic evidence, either from mutant or transgenic studies, has suggested their involvement in root development (see Supplementary Table S2 at JXB online). These genes were further classified based on their involvement in the growth and development of mature roots (primary roots and/or emerged LRs) and/or on LR formation (LR initiation and/or emergence).

Our in silico analysis revealed that, from the list of 98 RDR genes, 28% showed an altered expression in response to the nutrient deficiencies tested (Fig. 2). From these, 75% responded specifically to the deficiency of one nutrient. This observation supports the notion that the plant nutritional status alters RSA, primarily by modulating specific developmental steps. Noteworthy, none of those genes up- or down-regulated by more than one nutrient deficiency was associated with other factors, such as plant cultivation method or plant age (see Supplementary Table S1 and Supplementary Fig. S1 at JXB online). Since only two genes (XPL1 and ACR4) were significantly affected by the K-deficiency conditions tested (see Supplementary Fig. S1 at JXB online), K experiments were excluded from further analyses.

In the case of P, the analysis revealed that most RDR genes which were up- or down-regulated by P deficiency are related to LR formation (Fig. 2), such as PUCHI (Hirota et al., 2007) and WEI2/ASA1 (Sun et al., 2009). PUCHI regulates cell divisions during the establishment of LR primordia (Hirota et al., 2007), whereas WEI2/ASA1 is involved in jasmonic acid-dependent auxin synthesis promoting LR formation (Sun et al., 2009). This result is particularly interesting because LR density increases in response to low P availability (Williamson et al., 2001; Lopez-Bucio et al., 2002; Sanchez-Calderon et al., 2005; Gruber et al., 2013). Thus, in particular, the architectural modification of LR traits appears to be transcriptionally regulated by P deficiency. However, it is noteworthy that low P not only stimulates LR formation, but also arrests primary root elongation. The over-representation of RDR genes related to LR formation might therefore suggest that the induction of LR formation by P deficiency is more strongly transcriptionally regulated at the developmental level than the inhibition of primary root growth. Although provocative, this hypothesis may find support in other studies. Firstly, it has been shown that the stimulation of LR formation in P-deficient plants is independent of the primary root arrest (Perez-Torres et al., 2008). Secondly, the extent of the primary root inhibition under low P depends on the Fe levels available in the growth medium (Ward et al., 2008; Ticconi et al., 2009). In fact, when Fe is removed from a P-depleted medium, primary root elongation is restored. In addition, Fe concentrations increased in roots of P-deficient plants (Ward et al., 2008). Since Fe is toxic at elevated levels, an over-accumulation of this metal, for example, in the root meristem, could negatively affect cell biological processes. Unexpectedly, our analysis did not indicate that TIR1 was up-regulated by P deficiency, as shown in a previous study (Perez-Torres et al., 2008). However, it is noteworthy that the P deficiency-induced TIR1 expression is mainly confined to the central cylinder of roots and is there elevated by less than 2-fold. It is therefore likely that, within the experiments available in Genevestigator, this response was not detectable because whole root samples were collected and plants were older than those assessed by Perez-Torres et al. (2008).

In contrast to P, our analysis revealed that N deficiency also alters the expression of RDR genes involved in the development of mature roots (Fig. 2). In fact, mild N deficiency mainly alters the length of primary and lateral roots rather than LR density (Lopez-Bucio et al., 2003; Gruber et al., 2013). Thus, some of the RDR genes regulated by N could be involved in this RSA modification by affecting cell division rates in the meristem or the elongation of differentiated cells. In support of this view our survey indicated that N deficiency induces the expression of the cell wall-associated serine/threonine kinase WAK4 (Fig. 2), which affects root growth by regulating cell elongation (Lally et al., 2001). In addition, N deficiency up-regulates the expression of the shootward auxin transporter MDR4/PGP4 (Fig. 2). Since primary root length is significantly repressed in pgp4-1 mutants (Terasaka et al., 2005), MDR4 could be involved in the induction of primary and LR elongation under mild N deficiency. It is of note that, under severe N deficiency, LR formation is almost completely repressed (Krouk et al., 2010b; Gruber et al., 2013). Thus, the down-regulation of genes involved in LR formation, such as ACR4 and AXR5 (Yang et al., 2004; De Smet et al., 2008) could suggest that these genes are targeted during the repression of LR formation under severe N deficiency.

Similar to P deficiency, our analysis revealed that most of the RDR genes regulated by S limitation have assigned functions in LR formation (Fig. 2). This is particularly interesting as S deficiency induced relatively minor changes on the RSA compared with the changes under P deficiency (Dan et al., 2007; Gruber et al., 2013). By contrast, in older plants, Kutz et al. (2002) found that limiting S concentrations stimulate the initiation of LRs. Our expression analysis might support such a modulation. However, without knowledge on the RSA of the plants grown for the microarray analyses used here, it is not possible to accurately associate the changes with a characteristic change in root growth. In addition, S deficiency down-regulated the expression of SHY2 (Fig. 2), a gene that is involved in the regulation of cell differentiation in the root meristem (Tian and Reed, 1999). However, it remains to be tested whether a SHY2-dependent function is involved in the primary root response to S starvation (Fig. 1; Gruber et al., 2013).

Our in silico approach revealed that Fe deficiency altered the expression of RDR genes especially involved in LR formation (Fig. 2). In fact, LR density is decreased even by mild Fe deficiency (Gruber et al., 2013). Among the RDR genes responding to Fe deficiency, about 40% are related to auxin, including the auxin receptor TIR1, the auxin transporters PIN4 and MDR4, and the auxin-responsive genes LBD16 and LBD29 (Fig. 2). This effect is particularly interesting because both LBD16 and LBD29 act downstream from an auxin signalling cascade that controls LR formation (Okushima et al., 2007). The over-representation of auxin genes among those RDR genes regulated by Fe deficiency is also in agreement with recent studies. In fact, it has been shown that auxin is involved in many responses to Fe deficiency, such as the up-regulation of ferric-chelate reductase or H⁺-ATPase (Chen et al., 2010; Bacaicoa et al., 2011) and the induction of LR elongation in Fe-enriched patches (Giehl et al., 2012a). As observed for S deficiency, the expression of SHY2 was also down-regulated by Fe deficiency (Fig. 2). Since mild Fe deficiency also stimulated primary root elongation (Fig. 1; Gruber et al., 2013), it remains to be determined if SHY2-dependent modulation of cell differentiation is involved in this root architectural modification.

Concluding remarks

Recent progress in the quantitative analysis of different root traits from a single scanned image has developed a complex picture on the RSA changes evoked by different endogenous and exogenous triggers. Among the endogenous triggers, the plant nutritional status exerts a major impact on individual RSA traits that results in an impressive degree of root plasticity. On top of that, local nutrient availabilities can stimulate individual root developmental processes. Early morphological responses to varying nutritional conditions are based on the integration of local and systemic nutritional signals at specific steps or processes in root development. Under progressing nutrient deficiencies, nutritional functions referring to the maintenance of biomass may prevail.

So far, it is unclear why the availability of certain nutrients triggers changes in specific root traits while other nutrients do not, and whether these changes provide an adaptive advantage for plant performance or plant survival. At least, differences in the mobility of individual nutrients in the soil cannot explain common or distinct nutritional effects on RSA. More recent experiments indicate the existence of a sensing and signalling network that integrates information on the plant nutritional status and the external and spatial availability of nutrients into the developmental programme of the root. Uncovering the entry points of nutritional signals into the developmental programme may profit from systematic investigations carried out under comparable conditions for which expression analyses are also accompanied by RSA measurements. In addition, using mutants defective in root developmental genes for an assessment of nutrient-dependent changes in RSA traits may confirm specific or common genes and processes by which nutrients alter RSA. Ultimately, a deeper understanding of these processes will impact on nutrient placement strategies and crop breeding efforts for agricultural plant production that better exploit fertilizer nutrients and soil nutrient reserves by adaptive responses of crop root systems.