Bernadette B Banrezes

Thierry Sainte-Beuve

Eugénie Canon

Richard M Schultz

José-Manuel Cancela

Jean Pierre

Jean-Pierre P Ozil
email: jean-pierre.ozil@jouy.inra.fr

Euge ´nie Canon

Jose ´cancela

Adult body weight is programmed by a redox-regulated and energy-dependent process during the pronuclear stage in mouse

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Introduction

Fertilized mammalian eggs are sensitive to perturbations in metabolism that can become manifest following birth [START_REF] Ecker | Long-term effects of culture of preimplantation mouse embryos on behavior[END_REF][START_REF] Kwong | Support for Barker hypothesis upheld in rat model of maternal undernutrition during the preimplantation period: application of integrated 'random effects' statistical model[END_REF][START_REF] Young | Large offspring syndrome in cattle and sheep[END_REF], but the linkage between early quantitative changes in metabolism and specific long-term effects is poorly understood. Fertilization triggers a series of repetitive Ca 2+ oscillations that last for several hours and cease around the time of pronuclear (PN) formation [START_REF] Marangos | Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei[END_REF][START_REF] Miyazaki | Calcium signalling during mammalian fertilization[END_REF]. The regime of Ca 2+ signals drives a temporal set of events that constitute egg activation, including cortical granule exocytosis, cell cycle resumption, and dynamics of PN formation [START_REF] Ducibella | Egg-toembryo transition is driven by differential responses to Ca(2+) oscillation number[END_REF][START_REF] Nixon | Ca2+ oscillations and the cell cycle at fertilisation of mammalian and ascidian eggs[END_REF][START_REF] Toth | Egg activation is the result of calcium signal summation in the mouse[END_REF]. Manipulating the regime of Ca 2+ oscillations can result in longterm effects, e.g., altering the transcriptome in blastocysts and size of offspring [START_REF] Rogers | The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality[END_REF][START_REF] Ozil | Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term[END_REF]. Such findings provide further support that the Developmental Origins of Health and Disease hypothesis (DOHaD) [START_REF] Hales | The thrifty phenotype hypothesis[END_REF] can be extrapolated back to the initial period following fertilization when the egg metabolism is set off [START_REF] Dumollard | Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production[END_REF].

In various mammalian species different types of in-vitro stress can compromise the survival rate [START_REF] Zander | Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages[END_REF] and lead to either a reduction [START_REF] Khosla | Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes[END_REF] or increase in fetal growth [START_REF] Young | Large offspring syndrome in cattle and sheep[END_REF]. However, if stress affects embryo viability, it remains unknown whether metabolic perturbations during fertilization could impact post-natal growth of offspring. This study was designed to ascertain whether postnatal growth rate could be experimentally manipulated by selectively varying egg metabolism. Such a linkage between early egg metabolism and post-natal phenotype would provide an important experimental model for understanding DOHaD. Here we focus on two metabolic parameters i.e., the cellular redox state (NAD(P)H) and mitochondrial activity (FAD 2+ ), which are linked to the regime of Ca 2+ oscillations triggered by fertilization [START_REF] Dumollard | Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production[END_REF].

The intracellular redox state describes a complex interaction of the relative concentration of reduced and oxidized forms of a variety of molecules including the nicotinamide adenine nucleotides (NAD(P) + /NAD(P)H) and flavins (FAD 2+ /FADH 2 ). NAD(P)H electron carriers can impact gene expression through the action of several metabolic sensors, e.g., the Carboxyl-terminal Binding Proteins (CtBPs) [START_REF] Chinnadurai | CtBP, an unconventional transcriptional corepressor in development and oncogenesis[END_REF] or Sirtuins (SIRT) [START_REF] Mostoslavsky | At the crossroad of lifespan, calorie restriction, chromatin and disease: Meeting on sirtuins[END_REF][START_REF] Vaquero | The conserved role of sirtuins in chromatin regulation[END_REF], which can effect chromatin structure [START_REF] Zhang | SIRT1-dependent regulation of chromatin and transcription: Linking NAD(+) metabolism and signaling to the control of cellular functions[END_REF]. Variations in the redox potential may play an important role in regulating developmental processes in plants and metazoans [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF][START_REF] Allen | Redox control of transcription: sensors, response regulators, activators and repressors[END_REF][START_REF] Coffman | Mitochondria and metazoan epigenesis[END_REF] but the relationship between redox state in mammalian eggs and development remains unknown.

Following fertilization, the dynamics of Ca 2+ oscillations are linked to energy metabolism by stimulating mitochondrial ATP production via oxidative phosphorylation, and indeed, egg activation is accompanied by an increase in intracellular ATP concentration [START_REF] Dumollard | Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes[END_REF]. Ca 2+ released into the cytoplasm enters the mitochondrial matrix and activates the TCA cycle. During oxidative phosphorylation, a series of redox reactions transfers electrons initially from nutrients to NAD + /FAD 2+ , with the reduced forms in turn donating electrons to electron transport chain carriers located in the inner mitochondrial membrane, the final electron acceptor being oxygen [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF]. The energy harvested during these redox reactions is stored in an electrical and proton gradient across the inner mitochondrial membrane that drives ATP formation [START_REF] Vanblerkom | Mitochondria in early mammalian development[END_REF]. Mitochondrial ATP production in turn is essential to sustain the Ca 2+ oscillations that trigger events of egg activation [START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF].

Fertilized eggs can use exogenous pyruvate or lactate as an energy source; pyruvate serves as a cytoplasmic oxidant and mitochondrial reducing agent whereas lactate serves as a cytoplasmic reducing agent. NADH-mediated reduction of pyruvate consumes a proton, whereas NAD + -mediated oxidation of lactate generates a proton, and therefore is linked to intracellular pH (pH i ). Thus, modest fluctuations in the exogenous pyruvate/lactate ratio or pH can alter the energetic status and cytosolic redox state simultaneously, each of which can alter viability and fetal growth [START_REF] Mitchell | Disruption of mitochondrial malate-aspartate shuttle activity in mouse blastocysts impairs viability and fetal growth[END_REF].

To establish a connection between redox state in zygotes and the consequences on developmental outcomes following birth, it is essential that the perturbation has minimal effect on development to term. We exploited our ability to drive precisely and specifically the ''redox'' profile and mitochondrial activity by changing the lactate/pyruvate ratio or pH i of the culture medium [START_REF] Vanblerkom | Mitochondria in early mammalian development[END_REF][START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF][START_REF] Leese | Metabolic control during preimplantation mammalian development[END_REF] during the one cell stage with virtually no impact on development to term (see Results for further description of experimental rationale). We report here for the first time that post-natal growth rates can be up or down regulated by experimentally manipulating NAD(P)H and FAD 2+ signaling as early as the PN stage following fertilization.

Results

Experimental Rationale and Design

By exposing fertilized eggs to exogenous pyruvate, lactate or pH we experimentally manipulated the redox state of the cell, but only within a limited time period during which the fertilized egg partially uses its own stores of energy (e.g., amino acids, carbohydrates, fatty acids). To assess the potential role of pyruvate and lactate we chose a simple M16 medium over a more complex medium, e.g., KSOM/AA to maximize the impact of these treatments on the redox state. We then observed how a transient alteration of the redox state governed by a single exogenous carbohydrate, impacted growth and body weight of adult animals that developed from the manipulated fertilized egg, all other experimental parameters being equal.

We used NAD(P)H and FAD 2+ auto-fluorescence as a proxy for metabolic changes [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF][START_REF] Lane | Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo[END_REF][START_REF] Duchen | Imaging mitochondrial function in intact cells[END_REF], and designed four culture media with different carbohydrate compositions and pH to vary preferentially the mitochondrial activity (FAD 2+ signal) and the redox state (NAD(P)H signal). A micro-fluidic approach was employed to record precisely the kinetics of the auto-fluorescence signal during the PN stage. The treated embryos were transferred into foster mothers at the 2-cell stage and the growth profile of the offspring was determined until the 22 nd week of age as a phenotypical trait that reveals the long-term influence of egg metabolism.

Energy starvation causes NAD(P)H oxidation and low mitochondrial activity, and offspring, which are small before weaning, tend to catch up thereafter In mouse eggs, protein degradation accelerates shortly after fertilization and can provide a degree of energy autonomy for a few hours [START_REF] Tsukamoto | Autophagy is essential for preimplantation development of mouse embryos[END_REF][START_REF] Ferguson | A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development[END_REF]. Therefore, we first evaluated how fertilized eggs survived and developed to term following a 10 or 15 h period of starvation during the PN stage. We then correlated the egg NAD(P)H and FAD 2+ profiles with the growth profiles of the offspring.

Depleting exogenous carbohydrates induced a biphasic change of the NAD(P)H signal (Fig. 1A, blue line). This biphasic signal revealed that a virtually instant replacement of M16 by M starv induced an initial increase of NAD(P)H fluorescence followed by a rapid decline when intracellular carbohydrates were totally washed out. The increase in the FAD 2+ signal, which is of mitochondrial origin [START_REF] Dumollard | Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production[END_REF] (Fig. 1A, red line) and simultaneously monitored, suggested that mitochondrial activity, i.e., ATP production, decreased. During starvation for 10 or 15 h, both NAD(P)H and FADH 2 became more oxidized (Fig. S1). The transient change in pH i was due to sodium/proton equilibration due to replacing sodium lactate/pyruvate and glucose with NaCl to maintain osmolarity (Fig. 1A, green line).

Despite an elevated rate of NAD(P)H and FADH 2 oxidation, the incidence of cleavage to the 2-cell stage after 10 h of starvation was 65.2%, and 57.1% of embryos developed to term after embryo transfer (Table 1). In contrast, after 15 h of starvation, only 10.2% zygotes cleaved to the 2-cell stage, and of those only 30.7% developed to term (Table 1). Hence, the window of energy autonomy progressively declined between 10 and 15 h of starvation.

Both male and female offspring derived from zygotes starved for 10 h were significantly smaller (dark pink lines) than controls (black lines) by the time of weaning (Fig. 1B). The impact of litter size was evaluated by standardizing the data using the Z-score method (normalized data, see Materials and Methods). For males, the normalized growth profiles (Z-score) of experimental and control animals issued from the smaller litter sizes were similar, i.e., the highest limits of the shaded dark pink (experimental) and grey (control) zones were similar (Fig. 1C). In contrast, the Z-score profiles of experimental animals derived from larger litter sizes were below that of the controls, i.e., the lowest limit of the shaded dark pink zone (experimental) was beneath the lowest limit of the grey zone (control) (Fig. 1C). For females, the Z-score growth profiles although of smaller amplitude, display similar tendency (Fig. S2A). The effects induced by larger litter sizes during pregnancy amplified this negative effect on weight gain relative to control for both males and females (relative weight gain: RWG, Figs. 1D andS2B).

Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity and offspring are persistently smaller after weaning

To discriminate further whether high oxidation of cytosolic NAD(P)H or low mitochondrial activity (high FADH 2 oxidation) was responsible for the low post-natal growth, we cultured fertilized eggs for 15 h (the limit of energy autonomy) during which pyruvate was the sole energy source; pyruvate stimulates mitochondrial activity (i.e., maintaining a balanced FADH 2 / FAD 2+ ratio) but oxidizes the cytosolic NAD(P)H [START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF] as does starvation.

In mouse eggs, pyruvate serves as the main metabolite oxidized inside mitochondria for energy production, but is also reduced in the cytosol by the lactate dehydrogenase (LDH) with concomitant oxidation of NADH [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF][START_REF] Dumollard | Regulation of cytosolic and mitochondrial ATP levels in mouse eggs and zygotes[END_REF][START_REF] Lane | Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption[END_REF]. Pyruvate as the sole exogenous source of carbohydrate, however, may compromise pre-implantation development [START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF]. Accordingly, we evaluated survival to term and post-natal impact when fertilized eggs were exposed for only 15 h to the cytosolic NADH oxidant action of pyruvate.

Pyruvate alone caused an immediate decrease of the NAD(P)H signal (Fig. 2A, blue line), whereas the FAD 2+ signal (Fig. 2A, red line) remained unchanged as was pH i (Fig. 2A, green line). After 15 h in M pyr , the level of NAD(P)H oxidation remained low and the FAD 2+ signal remained at the resting level (Fig. S3). Hence, pyruvate supported a higher rate of cytosolic NAD(P)H oxidation and mitochondrial activity was not impaired by prolonged culture (15 h).

All treated fertilized eggs cleaved to the two-cell stage and following embryo transfer developed to term at a high incidence (82.1%, Table 1). Nevertheless, the post-natal growth profiles of animals of both genders (red lines) were significantly smaller than controls (back lines) but with the males also significantly smaller than those issued from starvation (dark pink lines) (Fig. 2B). The animals were healthy but remained small. This effect did not appear related to litter size because whatever the litter size, the normalized growth profiles (Z-score) of the higher and lower limits of the shaded red zone (experimental) remained below the upper and lower limits of the grey zone (control) (Fig. 2C for males and Fig. S4A for females). Hence, contrary to ''Starvation,'' whatever the litter sizes, the RWG following ''Pyruvate'' remained persistently low for both genders (Fig. 2D andS4B).

Exogenous lactate stimulates NAD + reduction and impairs mitochondrial activity, and offspring that are small at weaning catch up afterward To address whether the aforementioned phenotype could be changed by treatments that reverse the redox potential of the NAD + during the same period of time, we next cultured fertilized eggs in medium in which the sole energy source was lactate. In contrast to pyruvate, lactate is poorly metabolized by the mitochondria but is a strong cytosolic reductant via LDH activity [START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF]. Hence, we evaluated how offspring growth would be affected by a high rate of NAD + reduction and lactate metabolism within the 15 h time frame.

Replacing M16 with M lac induced an abrupt and simultaneous steep increase of NAD(P)H and FAD 2+ signals indicating that mitochondrial activity was virtually instantly down-regulated in the presence of lactate (Fig. 3A, blue and red lines); note that pH i was not altered by the treatment (Fig. 3A, green line). These two signals remained elevated at a constant level for 15 h (Fig. S5). Although the FAD 2+ signal indicated that mitochondrial activity was impaired during the treatment, almost all of the fertilized eggs cleaved to the 2-cell stage (Table 1). In addition, the high incidence of survival to term (73.4%, Table 1) suggests that lactate provided an additional source of energy that may or may not be related to some level of lactate-derived pyruvate production [START_REF] Dumollard | Regulation of redox metabolism in the mouse oocyte and embryo[END_REF]. Nevertheless, the growth of males (but not females) was significantly lower (blues lines) than that of the controls (black lines) but significantly higher than that following the ''Pyruvate'' treatment (red lines) (Fig. 3B,blue6symbol). The normalized growth profiles (Z-score) of experimental and control animals derived from the group of smaller litter sizes (5, 4, 3 and 2) were very similar, i.e., the upper limits of the shaded blue (experimental) and grey (control) zones overlap before weaning and at adult age (Fig. 3C for males and Fig. S6A for females). In contrast, the Z-score profiles of experimental male animals derived from the group of larger litter sizes (8, 7 and 6) were below those of the controls, i.e., the lowest limit of the stippled blue zone (experimental) was beneath the lowest limit of the grey zone (control) (Fig. 3C). No litter size effect was observed for the females (Figs. S6A andB). For males, this effect appeared related to litter size because the RWG of males derived from smaller litter sizes was higher than that for males derived from larger litter sizes (Fig. 3D). Unlike the ''Pyruvate'' treatment, larger litter sizes during pregnancy have a tendency to amplify a negative effect of ''Lactate'' at least for the males. The growth profiles for females were similar to those of the controls but significantly higher than ''Pyruvate'' (red line) (Fig. 3B, blue+symbol). Hence, elevated NAD + reduction caused by lactate reversed the tendency observed with high oxidation by pyruvate for both genders but without reaching that of the control.

Cytosolic alkalization increases NAD(P) + reduction and offspring that are normal at birth become persistently large after weaning

To circumvent the impact on mitochondrial activity when lactate was the sole energy source, we developed a procedure that consisted of first culturing fertilized eggs in standard M16 culture media to maintain the normal supply of exogenous carbohydrates, and then transiently increased the external pH to down-regulate the oxidative activity of the mitochondria by dissipating the proton gradient outside the mitochondrion matrix that is known to correlate with NAD(P)H increase in sea urchin eggs [START_REF] Schomer | Redox changes during fertilization and maturation of marine invertebrate eggs[END_REF][START_REF] Whitaker | The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs[END_REF].

Fertilized eggs cultured in M16 culture media loaded with NaOH, which resulted in a pH value of 8.2, displayed steep increase in pH i (Fig. 4A, green line) that reached a plateau within a few minutes. The NAD(P)H signal (Fig. 4A, blue line) increased virtually instantaneously whereas the FAD 2+ signal (Fig. 4A, red line) remained essentially unchanged, showing that the balance between reduction and oxidation of FADH 2 /FAD 2+ inside the mitochondrion matrix remained balanced at a high pH i .

The pH i -dependent rise in NAD(P)H was similar to that observed in sea urchin eggs following fertilization [START_REF] Schomer | Redox changes during fertilization and maturation of marine invertebrate eggs[END_REF][START_REF] Whitaker | The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs[END_REF]. The kinetics of the proton/hydroxide equilibrium between the outside and inside of the fertilized egg was very fast and highly reversible (Fig. 4A), and might involve translocation of protons across the plasma membrane [START_REF] Deamer | Proton permeation of lipid bilayers[END_REF] that bypasses the fertilized egg's normal regulative processes [START_REF] Amirand | Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis[END_REF][START_REF] Lane | Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter[END_REF][START_REF] Phillips | Intracellular pH change does not accompany egg activation in the mouse[END_REF]. Although we cannot dissociate the impact of pH i from the changes in NAD(P)H, the NAD(P)H signal did not remain high, but decreased within 3-5 h to the resting level (Fig. S7). Under these conditions, all of the fertilized eggs cleaved to the 2cell stage and 66.7% of them developed to term (Table 1). These results demonstrate that a transient rise in pH i and NAD(P)H during the PN stage did not compromise development to term but the growth of both genders (green lines) was significantly accelerated immediately after birth in comparison to the controls or to ''Pyruvate'' (red lines) (Figs. 4B andC). This effect did not appear related to litter size because after weaning, whatever the litter size, the normalized growth profiles (Z-score) of the upper and lower limits of the shaded green zone (experimental) remained above the highest limit of the grey zone (control) (Fig. 4C for males and Fig. S8A for females). Like the ''Pyruvate'' treatment, but unlike the ''Starvation'' and ''Lactate'' treatments, the RWG was not affected by litter size for both genders (Figs. 4D andS8B).

Discussion

We report here that experimentally manipulating the redox potential in fertilized eggs during the PN stage does not compromise viability but has long-term consequences that become manifest on post-natal growth rates (summarized in Fig. 5). All else being equal-embryo production, pregnancy length restricted to 19 days, and animal breeding-when egg mitochondrial activity is compromised by the absence of exogenous pyruvate, i.e., with ''Starvation'' and ''Lactate'' treatments, eggs in both cases develop to term but the offspring display a smaller post-natal growth rate than ''Control'', mostly for the males (Figs 1B and3B). Thus, eggs can survive a transient degree of energetic depression during the PN stage (Table 1) but at the cost of post-natal growth. Moreover, the high or low NAD(P)H redox level imposed by ''Starvation'' or ''Lactate'' does not appear to have long-term consequences because the growth of animals tends to catch up whatever the redox potential (Figs 1 and3). In contrast, when mitochondrial activity is supported by pyruvate in the culture media, post-natal growth profile varies as a function of high or low redox potential. When cytosolic NAD(P)H oxidation is set high (low reducing power) by ''Pyruvate'' treatment the adults (males and females) are smaller, whereas when cytosolic NAD(P)H oxidation is set low (high reducing power) by transient cellular alkalization the growth rate accelerates and results in larger adult animals (see Figs. 4B and4D where the growth profiles of ''Pyruvate'', ''Control'' and ''Alkalization'' are superimposed). Because the experimental design provides a high survival rate to term (Table 1) it is possible to observe a strong linkage between transient changes in egg metabolism (redox and mitochondrial activity) and post-natal development. Our finding that male and female offspring display different levels of sensitivity to the treatments presumably reflects differences in their chromosomal complement and physiology [START_REF] Gardner | Sex-related physiology of the preimplantation embryo[END_REF]. The present results underscore previous findings that the gender difference is an intriguing feature of developmental programming [START_REF] Watkins | Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring[END_REF].

We also find that when egg metabolism is compromised by ''Starvation'' and ''Lactate'' treatments, the growth profile in both cases depends on litter size. Mice from smaller litter sizes have a normal RWG whereas mice from larger litter sizes have a smaller RWG (Figs. 1D and3D). Thus, when maternal resources per fetus are sufficient during fetal life (i.e., small litter size), eggs experiencing ''Starvation'' or ''Lactate'' treatments recuperate and a normal growth rate occurs during their fetal life. Thus, a transient metabolic impact during the PN stage is reversible and sensitive to a litter size effect during fetal life whatever the redox potential during the treatment. In contrast, when egg mitochon- drial activity is not impaired by the absence of exogenous pyruvate, the growth profile is insensitive to litter size whatever the redox potential. With ''Pyruvate'' treatment, mice from either smaller or larger litter sizes have a smaller RWG (Figs. 2D andS4B), whereas with the ''Alkalization'' treatment, mice from lower or higher litter sizes both have a higher RWG (Figs. 4D andS8B). Thus, when egg mitochondrial activity is not impaired during fertilization, the impact on the RWG is not prone to changes in relation to the maternal resources during pregnancy, i.e., litter size. In these experimental situations (''Pyruvate'' and ''Alkalization'' treatments), the long-term impact appears set at the PN stage and remains insensitive to litter size during fetal life, with a permanent impact of a high or low redox potential. The high or low redox states have different post-natal outcomes at the adult age depending upon mitochondrial activity that appears to be a key actor in setting either long or short-lasting responses. One simple hypothesis for this observation is that a highly sensitive metabolic sensor, which remains to be identified, translates the mitochondrial activity and modulates the long term responses as outlined in Fig. 5.

Starvation and lactate treatments lead to low mitochondrial activity of the egg that is impaired because the FAD 2+ signal immediately increases (FADH 2 becomes more oxidized) and stays high during the treatments (Figs. 1A and3A). That a high or low NADH redox potential results in similar post-natal growth (small offspring whose growth catches up later in life) indicates that low mitochondrial activity during the treatment overrides the potential long-term effect of the NAD(P)H redox potential. In contrast, pyruvate treatment does not compromise the mitochondrial activity but results in permanently small animals, whereas ''Starvation'', which also causes NADH oxidation, results in small animals that catch up after weaning.

It is likely that the ''Alkalization'' treatment at least partially impairs mitochondrial activity energy production by dissipating the mitochondrial proton gradient (Fig. 4A) [START_REF] Vanblerkom | Mitochondria in early mammalian development[END_REF]. Nevertheless, the energy status of the cell is likely to be far less compromised than for the starvation treatment since the survival rate at the 2-cell stage is identical to the controls but higher than those issued from starvation (Table 1). This is probably due to the presence of exogenous pyruvate during alkalization that helps the egg to cover its energy demand during the treatment and the short duration of the mitochondrial perturbation due to the rapid restoration of the resting pH i level by the buffering action of bicarbonate in the presence of CO 2 (see Fig. S7). In this situation, the transient increase in NAD(P)H signal (high reducing power) is capable of accelerating post-natal growth. Moreover, the intensity of the postnatal growth suggests that the treatment requires only a short period of a few hours to provoke a long lasting impact.

The results described here also provide further evidence that post-natal weight variation is sensitive to changes in the Ca 2+ regime at fertilization [START_REF] Ozil | Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse[END_REF], which increase mitochondrial activity and thus augments reducing equivalents of NADH and FADH 2 [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF]. Our findings support a previous hypothesis suggesting that an electrochemical sensor(s) is capable of influencing developmental processes according to small changes in egg metabolism at the time of fertilization [START_REF] Dumollard | The role of mitochondrial function in the oocyte and embryo[END_REF][START_REF] Blackstone | Metabolic gradients: a new system for old questions[END_REF][START_REF] Coffman | Mitochondrial patterns and function in animal development[END_REF]. At this juncture, however, it is difficult to ascribe a molecular mechanism that underpins the observed changes in postnatal growth rate. An obvious candidate is failure to recapitulate faithfully the dramatic reprogramming of gene expression during the 2-cell stage; such reprogramming is essential for further development [START_REF] Schultz | The molecular foundations of the maternal to zygotic transition in the preimplantation embryo[END_REF]. We have conducted microarray experiments on 2-cell embryos derived from controls and manipulated 1-cell embryos. An unsupervised cluster analysis did not reveal any difference between the control and experimental groups. Moreover, of the few mis-expressed transcripts, the change in relative abundance of affected transcripts was typically within 20% of the control. Finding that reprogramming occurs with relatively high fidelity in the treated embryos is not surprising because we find development to term is unaffected, whereas substantially perturbing the fidelity of reprogramming leads to either developmental arrest at the 2-cell stage or a marked delay in preimplantation development [START_REF] Bultman | Maternal BRG1 regulates zygotic genome activation in the mouse[END_REF].

By manipulating the metabolism of a fertilized egg without perturbing survival to term we have unveiled a surprising linkage between a very brief perturbation in metabolism of a 1-cell embryo and changes in adult phenotype. Exploiting this property has obvious practical applications in animal reproductive biotechnology. The results also have clinical implications regarding use of assisted reproductive technologies (ART) to treat human infertility. Embryo culture commencing at the 1-cell stage is an intrinsic part of ART. Previous studies using a mouse model predicted several years in advance the observed increased risk for ART-conceived children for loss-of-imprinting syndromes [e.g., [START_REF] Watkins | Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring[END_REF][START_REF] Ozil | Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse[END_REF].

Materials and Methods

Ethics Statement

Embryo production and collection

Female mice (C57BL/66CBA) were superovulated by i.p. injection of 8 IU of PMSG (Syncro-PartH CEVA) followed 48 h later by injection of 7.5 IU of hCG (ChorulonH Intervet) and caged overnight with F1 males. Fertilized eggs were recovered 17 h after hCG injection, and then placed in drops under mineral oil and incubated under an atmosphere of 5% CO 2 in air at 37uC. Only eggs that displayed two PN were subjected to experimental treatments.

Culture media and embryo treatment

Culture media were formulated from standard M16 that has the highest lactate to pyruvate ratio [START_REF] Whittingham | Culture of mouse ova 80[END_REF]. Variation of the NAD(P)H and FAD redox levels was obtained by using a single source of carbohydrate in the culture medium at standard concentration, i.e., 23.28 mM lactate or 0.33 mM pyruvate, which are referred to as M lac and M pyr , respectively. Starvation experiments were performed with M16 medium devoid of carbohydrates (M starv ). The effect of extracellular pH was explored by adding 2 mM NaOH into the M16 medium containing the normal composition of carbohydrates (M pH ). NaCl concentration was increased in M starv , M lac and M pyr to maintain osmolarity. All chemicals were purchased from Sigma and VWR. Fertilized eggs were washed thoroughly and placed in 50 ml drops of experimental media (10-30 embryos per drop) under paraffin oil and were cultured for 10 or 15 h in an atmosphere of 5% CO 2 in air at 37uC. The 2-cell embryos were then washed in M16 and transferred to pseudo-pregnant recipients. Four experiments and one control were designed. Treatment ''Starvation''; eggs were cultured in M starv for 10 or 15 h. Treatment ''Pyruvate''; eggs were cultured in M pyr for 15 h. Treatment ''Lactate''; eggs were cultured in M lac for 15 h. Treatment ''Alkalization''; eggs were cultured in M pH for 15 h. Treatment ''Control''; eggs were cultured in M16 for 15 h.

Intracellular fluorescence recording

Two eggs were held individually in a microfluidic chamber by two holding micropipettes in a viewing field of a Nikon TE2000 inverted microscope fitted with a Fluor 406 oil immersion objective (NIKON) as previously described [START_REF] Ozil | Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse[END_REF]. The optical field was illuminated with a 75-W Xenon arc lamp (OSRAM) and specific excitation wavelengths were selected from a Cairn Optoscan Monochromator (DIPSI). NAD(P)H autofluorescence was measured by excitation at 365 nm65 nm. The emission was collected through a band-pass filter (445-45 nm) (Semrock Filter). FAD 2+ autofluorescence was measured by excitation at 450 nm616 nm. The emission was collected at 520-35 nm. Fluorescence intensity was captured using the EMCCD QuantEM 521SC (5126512 pixel) cooled camera from Photometric (Roper Scientific) with 300 ms exposure time. The whole process was controlled by the MetaFluor software v7.1 (Roper Scientific). All records are the average value of 6 records and are plotted with SigmaPlot 11 software (Ritme Informatique). For measuring variation of pH i , eggs were incubated at 37uC for 15 min in M16 with 20 mM SNARF-1-AM (Invitrogen). SNARF-1 fluorescence was detected with an excitation wavelength of 535 nm and two emission wavelengths of 640 nm and 600 nm [START_REF] Duchen | Imaging mitochondrial function in intact cells[END_REF]. The pH i records are independent of the NAD(P)H/FAD 2+ records.

Microfluidic device and method

The microfluidic chamber previously described to drive Ca 2+ signaling [START_REF] Ozil | Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse[END_REF] makes possible the ability to change the entire chamber volume (10 ml) in less than 1 second at 37uC. All tubing is gas impermeable (Upchurch-Rheodyne). The media were loaded into the central unit (prototype BRACER; Jouy-en-Josas, France) directly from the incubator (5% CO 2 in air). Eggs were subjected to three sequences of microfluidic flux. The first sequence makes it possible to record the resting signals in M16; the second, the kinetic of changes induced by the experimental media for 15 min; and the third, the kinetic of changes when M16 is back into the chamber.

Embryo transfer and animal Phenotyping

Female F1 mice (C57BL/66CBA), 8-13 weeks old, were used as recipients. Experimental and control eggs at the two-cell stage were transferred in groups of 8 to the left oviduct of each recipient female. Starting the first week after delivery, newborns were weighed every week until the 22 nd week. The pups were weaned at the 4 th week and males and females in each litter were separated and placed in groups of 5 in independent cages. Mice were fed with a standard laboratory diet and tap water ad-libitum. We calculated the relative weight gain (RWG) as follows: for week n, RWGn is the difference between the mean weight of the experimental and mean weight of the control animals divided by the mean weight of the control animals.

Statistical analysis

The data were analyzed using SigmaStat 3.0 software. The Chisquare test was used to analyze the rate of cleavage to the two-cell stage, the survival rate at term, the gestation length, the mean litter (Table 1). Growth data were converted to Z-scores prior to statistical analysis. We used SigmaPlot V11 to standardize the entire data set (n = 210 living animals) to the same scale (m = 0, s = 1). Student's t test was used for comparing the Z values of the experimental groups versus the control. P,0.05 was considered to be statistically significant. To visualize the impact of litter size on growth better, we also separately plotted the Z-values of animals issued from the larger litter size which are above the average value of litter sizes (5.5 see Table 1) that include 8, 7 and 6 litter sizes, and from the lower litter sizes which are below the average value, i.e., 5, 4, 3 and 2 (only 2 females gave birth to 2 offspring in control and lactate experiments. None of the females delivered a single offspring). Significant differences between correlations were analyzed week by week. For all tests, sample numbers and P values are included in the text.

Figure 1 .

 1 Figure 1. Energy starvation and developmental responses. Metabolic profiles and post-natal phenotypes induced by ''starvation.'' (A) Time course of intracellular NAD(P)H (blue), FAD 2+ (red) signals (grey levels, g.l., 14 bits) and pH i fluorescence (green) of eggs subjected to M starv . The initial NAD(P)H biphasic curve is due to the high lactate/pyruvate ratio in M16 that delays intracellular depletion of carbohydrates. (B) Growth profiles of control (black lines) and experimental animals (dark pink lines). The data are expressed as mean 6 SEM. (C) Standardized plots of the total Z values (n = 210). The upper limits of the shaded zones (grey for controls and dark pink for the experimental values) are the normalized weight profiles of males issued from the larger litter sizes (8, 7 and 6) and the lower limits, from the smaller litter sizes (5, 4, 3 and 2). (D) Relative weight gains (RWG) of experimental animals according to the larger or smaller litter size group. In this figure and in figures 2-4, the stars in panels B and C denote significant differences between average experimental and control values (with a P-value at least ,0.04) when compared to controls. doi:10.1371/journal.pone.0029388.g001

Figure 2 .

 2 Figure 2. Exogenous pyruvate and developmental responses. Metabolic profiles and post-natal phenotypes induced by ''pyruvate.'' (A) Time course of intracellular NAD(P)H (blue), FAD 2+ (red) signal (grey levels, g.l., 14 bits) and pH i fluorescence (green) of eggs subjected to M pyr . (B) Growth profiles of control (black lines) and experimental (red lines) animals. The growth profiles of M starv animals from Fig. 1B are plotted as dark pink lines for comparison. The data are expressed as mean 6 SEM. (C) Standardized plots of total Z values (n = 210). The upper limits of the shaded zones (grey for the controls and red for the experimental values) are the normalized weight profiles of males issued from the larger litter sizes (8, 7 and 6) and the lower limits, from the smaller litter sizes (5, 4, 3 and 2). (D) Relative weight gains (RWG) of experimental animals according to the larger or smaller litter size group. doi:10.1371/journal.pone.0029388.g002

Figure 3 .

 3 Figure 3. Exogenous lactate and developmental responses. Metabolic profiles and post-natal phenotypes induced by ''Lactate.'' (A) Time course of intracellular NAD(P)H (blue), FAD 2+ (red) signals (grey levels, g.l., 14 bits) and pH i fluorescence (green) of eggs subjected to M lac . (B) Growth profiles of control and experimental animals. The growth profiles of M pyr animals from Fig. 2B are plotted as red lines for comparison. The blue crosses and plus symbols show when the M lac profiles are significantly higher than the M pyr profiles for males and females, respectively. The data are expressed as mean 6 SEM. (C) Standardized plots of total Z values (n = 210). The upper limits of the shaded zones (grey for the controls and blue for the experimental values) are the normalized weigh profiles of males issued from the larger litter sizes (8, 7 and 6) and the lower limits from the smaller litter sizes (5, 4, 3 and 2). (D) Relative weight gains (RWG) of experimental animals according to the larger or smaller litter size group. doi:10.1371/journal.pone.0029388.g003

Figure 4 .

 4 Figure 4. Alkalization and developmental responses. Metabolic profiles and post-natal phenotypes induced by ''alkalization.'' (A) Time course of intracellular NAD(P)H (blue), FAD 2+ (red) signals (grey levels, g.l., 14 bits) and pH i fluorescence (green) of eggs subjected to M pH . (B) Growth profiles of control and experimental animals. The growth profiles of M pyr animals from Fig. 2B are plotted as red lines for comparison. The data are expressed as mean 6 SEM. (C) Standardized plots of total Z values (n = 210). The upper limits of the shaded zones (grey for the controls and green for the experimental values) are the normalized weight profiles of males issued from the larger litter sizes (8, 7 and 6) and the lower limits from the smaller litter sizes (5, 4, 3 and 2). (D) Relative weight gains (RWG) of M pH experimental animals according to the larger or smaller litter size group (green lines). The RWG of M pyr animals from Fig. 2B are plotted as red lines for comparison. doi:10.1371/journal.pone.0029388.g004

Figure 5 .

 5 Figure 5. Post-natal outcomes resulting from zygotic metabolic sensor activities. The diagram represents the linkages between the composition of the culture media, the NAD(P)H and FAD 2+ concentrations and the adult phenotype, that are mediated by a zygotic metabolic sensor. doi:10.1371/journal.pone.0029388.g005

  Animal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals (Directive 86/609EC). All efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments of the author's institution, INRA (permit number 11 024).

Table 1 .

 1 Developmental potential of experimental and control eggs. The rate of two-cell of the M starv 10 and 15 hours are significantly lower than the other groups, Chi-square test (p,0.001). **The rate of survival of the M starv 15 hours are significantly lower than the pyruvate group, Chi-square test (p = 0.017).Dead newborns at week n: w(n). 8 eggs were transferred for every recipient except for the M starv 15 h group. doi:10.1371/journal.pone.0029388.t001

	Treatment	# Egg	# 2-C (%)	# Recipient	# 2-C transferred number a	# Pup	Survival (%)	Litter size±SEM
	Control	351	351 (100)	11	88	56 (1{w2)	63.6	5.160.5
	M starv 10 h	164	107 (65.2)*	7	56	32 (2{w1; w8)	57.1	4.660.9
	M starv 15 h	234	24 (10.2)*	2	13	4	30.7**	260.0
	M pyr	157	157 (100)	7	56	46	82.1	6.660.7
	M lac	179	177 (98.9)	8	64	47 (2{w1; w9)	73.4	5.960.4
	M pH	91	90 (98.9)	6	48	32 (2{w1; w3)	66.7	5.360.6
	Total	1176	907	41	325	217	66.7	5.560.3
	# = number.

1

* and ** denotes significant differences. *{

PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29388

PLoS ONE | www.plosone.org

December 2011 | Volume 6 | Issue 12 | e29388

Acknowledgments

The authors would like to thank Dr Alireza Fazeli and Donald White for their stimulating discussions and support. We thank Luc Reverbel and Je ´ro ˆme Pottier from the unit of Infectiologie Expe ´rimentale des Rongeurs et Poissons (UE IERP INRA-Jouy) for excellent animal care. The authors, except RMS, are members of the COST action FA0702 (www.cost-gemini. eu).

This research was supported by INRA (Animal Physiology and Livestock Systems Division) to JPO and grant from the NIH (HD022732) to RMS. BRACER BIOTECH supported the development of the microfluidic device and the software (contract B05806). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supporting Information

Author Contributions

Conceived and designed the experiments: J-PO. Performed the experiments: BB J-PO TS-B EC. Analyzed the data: J-PO BB JC RMS. Contributed reagents/materials/analysis tools: BB J-PO TS-B EC. Wrote the paper: J-PO JC RMS.


Ive De Smet

Philip J White

A Glyn Bengough

Lionel Dupuy

Boris Parizot

Ilda Casimiro

Renze Heidstra

Marta Laskowski

Marc Lepetit

Frank Hochholdinger

Xavier Draye

Hanma Zhang

Martin R Broadley

Benjamin Pe

John P Hammond

Hidehiro Fukaki

Sacha Mooney

Jonathan P Lynch

Phillipe Nacry

Ulrich Schurr

Laurent Laplaze

Philip Benfey

Tom Beeckman

Malcolm Bennett

Analyzing Lateral Root Development: How to Move Forward

Roots are important to plants for a wide variety of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface between the plant and various biotic and abiotic factors in the soil environment. Therefore, understanding the development and architecture of roots holds potential for the manipulation of root traits to improve the productivity and sustainability of agricultural systems and to better understand and manage natural ecosystems. While lateral root development is a traceable process along the primary root and different stages can be found along this longitudinal axis of time and development, root system architecture is complex and difficult to quantify. Here, we comment on assays to describe lateral root phenotypes and propose ways to move forward regarding the description of root system architecture, also considering crops and the environment.

Root system architecture is a key determinant of nutrient and water use efficiency and describes, on a macro scale, the organization of the primary root and root-and stemderived branches where they are present in monocots and dicots [START_REF] Hochholdinger | From weeds to crops: Genetic analysis of root development in cereals[END_REF][START_REF] De Smet | Lateral root initiation or the birth of a new meristem[END_REF][START_REF] Hochholdinger | Conserved and diverse mechanisms in root development[END_REF][START_REF] Pe ´ret | Arabidopsis lateral root development: An emerging story[END_REF][START_REF] Coudert | Genetic control of root development in rice, the model cereal[END_REF]. Overall root architecture of dicots, such as Arabidopsis thaliana, and monocots, such as maize (Zea mays) and rice (Oryza sativa), differs. The dependence on the embryonically derived system of a primary root and its lateral roots is a feature typical of dicots (taproot system). Perennial dicot species, for example, Brassica, usually have a broadly similar root system in terms of development and morphological layout to Arabidopsis, though their comparatively longer life cycles usually result in a larger and denser root system. In maize and rice, the postembryonic shoot-borne root system with many different types of root branches builds the majority of the root system (fibrous root system). On a micro scale, root system architecture includes root hairs that increase the surface area [START_REF] Gilroy | Through form to function: Root hair development and nutrient uptake[END_REF][START_REF] Tominaga-Wada | New insights into the mechanism of development of Arabidopsis root hairs and trichomes[END_REF]. In addition, adaptive root structures, such as cluster roots [START_REF] Neumann | Cluster roots-An underground adaptation for survival in extreme environments[END_REF][START_REF] Lambers | Root structure and functioning for efficient acquisition of phosphorus: Matching morphological and physiological traits[END_REF], nitrogen-fixing nodules [START_REF] Crespi | De novo organ formation from differentiated cells: Root nodule organogenesis[END_REF][START_REF] Oldroyd | Coordinating nodule morphogenesis with rhizobial infection in legumes[END_REF], and mycorrhizae [START_REF] Bonfante | Dating in the dark: How roots respond to fungal signals to establish arbuscular mycorrhizal symbiosis[END_REF][START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: New paradigms from cellular to ecosystem scales[END_REF], can improve water and nutrient uptake by the plant root system. Here, we will focus on lateral roots.

In the past, various approaches have been used for lateral root phenotyping, ranging from total numbers of (emerged) lateral roots, (emerged) lateral root density, lateral root index, etc. [START_REF] Dubrovsky | The lateral root initiation index: an integrative measure of primordium formation[END_REF]Dubrovsky and Forde, 2011). Dubrovsky and Forde (2011) suggested a set of methods to analyze lateral root development quantitatively in Arabidopsis. While Dubrovsky and Forde (2011) offer useful points for consideration to reduce mistakes describing root branching phenotypes, their solution is restricted in scope to Arabidopsis grown on agar. In addition, notwithstanding their parameters can have value for in-depth analyses of Arabidopsis root architecture, these approaches should not become the sole standard in phenotyping root branching. Also, with the growing importance of studying root system architecture in crops, including cereals, we require a wider portfolio of assays to address different aspects of root systems. With respect to (cereal) crop species, there are other features, such as volume, surface area, length distribution, etc., that are straightforward to measure and can be taken into account [START_REF] Iyer-Pascuzzi | Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems[END_REF]. Here, we further highlight novel and original approaches that move from two-to three-dimensional (2D to 3D) image analyses, and we phrase the importance for wider adoption of imaging approaches capturing the dynamic nature of roots, such as root branching kinetics and interactions with the environment. Moreover, we stress the future need to look at lateral root development in more ecologically and agronomically realistic contexts, such as when grown in soil or even under field conditions. All these aspects are important for both small-scale and highthroughput approaches, with the latter also being tackled in several phenotyping facilities.

WHAT MEASUREMENTS CAN WE USE?

Simple measurements of lateral root density (lateral root primordia and/or lateral roots per unit of total primary root length or of branching zone) remain useful statistics when interpreted appropriately and should be sufficient to characterize root branching phenotypes in the first instance. In addition, the Arabidopsis model system can be used to gain further insight into the developmental basis for an altered number of emerged lateral roots in given mutant backgrounds, and the total number and distribution of stages of lateral root primordia can be determined in wild-type versus mutant roots [START_REF] Swarup | The auxin influx carrier LAX3 promotes lateral root emergence[END_REF]. Nevertheless, such analyses are commonly performed in a static way (i.e., only taking one time point into account). While single time point assays can provide informative answers, the age of the plant is important as emergence of lateral roots varies considerably with time as suggested by the variable densities reported (Dubrovsky and Forde, 2011). In Arabidopsis, lateral root primordia emerge at ;7 d after germination, soon after the first pairs of leaves are visible as they represent the source of auxin to promote root outgrowth [START_REF] Bhalerao | Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings[END_REF]. Hence, small delays in (leaf) development can provoke major temporal differences in lateral root density that disappear in older plants. In addition, a naturally heterogenous environment affects root system architecture at a specific point in time and space. As such, the root system also reflects previous local environmental situations [START_REF] Fu ¨llner | Vertical gradient in soil temperature stimulates development and increases biomass accumulation in barley[END_REF]. Therefore, we should be mindful that time is an important parameter when considering development of primordia and root systems.

In addition, the importance of events occurring before the stage I primordia are visible has lately become more apparent in Arabidopsis [START_REF] De Smet | Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis[END_REF][START_REF] Moreno-Risueno | Oscillating gene expression determines competence for periodic Arabidopsis root branching[END_REF]. In this respect, the production of prebranch sites and founder cells are features that could also be taken into account when describing lateral root phenotypes. In Arabidopsis, this can be addressed using available markers, such as pDR5:LUCIFERASE [START_REF] Moreno-Risueno | Oscillating gene expression determines competence for periodic Arabidopsis root branching[END_REF]), pDR5:b-glucuronidase (De Smet et al., 2007), pDR5rev:green fluorescent protein [START_REF] Dubrovsky | Auxin acts as a local morphogenetic trigger to specify lateral root founder cells[END_REF], and pGA-TA23:nuclear localization signal:green fluorescent protein [START_REF] De Rybel | A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity[END_REF]. Besides how many lateral roots are being produced, it is also important to determine how fast they grow and how they are positioned along the primary root axis.

As current approaches neglect the fact that lateral root density changes as a function of the rate of root growth (Dubrovsky and Forde, 2011) and to avoid drawing incorrect conclusions based on variation in lateral root densities at one time point, we need to monitor the dynamics of lateral root development. To zoom in on a potential alteration of the process of lateral development and/or emergence, focused, reliable, and reproducible quantitative phenotyping based on manually curved roots (J-hooks) [START_REF] Laskowski | Root system architecture from coupling cell shape to auxin transport[END_REF] or the gravistimulation of lateral roots in a well-defined zone where the root apex has been reorientating its growth toward the new gravity vector are useful approaches [START_REF] Lucas | Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation[END_REF][START_REF] Guyomarc'h | Early development and gravitropic response of lateral roots in Arabidopsis thaliana[END_REF]. In the latter approach, subsequent stages of lateral root development occur at regular time points following gravistimulation, making this an ideal system to record specific deviations from the normal lateral root organogenesis process and for analyzing the timing of gene expression (De Smet et al., 2010).

WHICH GROWTH MEDIA AND CONDITIONS TO STUDY ROOTS?

To study roots, we need ways to culture and observe them in the lab. There is a large diversity of growth media reported in the literature, and Dubrovsky and Forde (2011) highlighted that this as a serious limitation to the robustness and repeatability of results across laboratories. However, when significant differences are reported on lateral root densities, and both mutants and the wild type were properly compared on the same medium, there should not be a problem interpreting the data. In many cases, the medium has little interaction with root responses to experimental treatments, and many mutants conserve their phenotypes across a range of environmental conditions. It is difficult to imagine how the scientific community could benefit from a standardization of growth media that would hinder our understanding of root plasticity. Especially since growth conditions are quite different between labs (temperature, light intensity, photoperiod, humidity, etc.) and since standardizing media would not be sufficient to create homogenous conditions. However, as also suggested by Dubrovsky and Forde (2011), a greater level of detail about growth conditions employed in a research article would certainly aid attempts to replicate experiments.

Adaptation of root system architecture to variable environments is highly plastic, and exploring the diversity of these developmental adaptations is one of our key challenges for the future. Root developmental strategies strongly depend on both local concentrations of nutrients as well as on the nutrient status of the whole plant [START_REF] Gojon | Root uptake regulation: A central process for NPS homeostasis in plants[END_REF] and on soil temperatures [START_REF] Fu ¨llner | Vertical gradient in soil temperature stimulates development and increases biomass accumulation in barley[END_REF]. We believe that the scientific community might not necessarily profit from the general use of one single growth medium, given that substantial progress has been realized using different media and different growth conditions, instead of one standardized medium. We cannot understand roots without knowing how they adapt to their environment. Experimental studies must therefore expose roots to heterogeneous physical or chemical environments. Split root systems [START_REF] Remans | The Arabidopsis NRT1.1 transporter participates in the signal-ing pathway triggering root colonization of nitrate-rich patches[END_REF][START_REF] Girin | Identification of Arabidopsis mutants impaired in the systemic regulation of root nitrate uptake by the nitrogen status of the plant[END_REF][START_REF] Ruffel | Nitrogen economics of root foraging: transitive closure of the nitrate-cytokinin relay and distinct systemic signaling for N supply vs. demand[END_REF] or artificial granular media [START_REF] Bengough | PIV as a method for quantifying root cell growth and particle displacement in confocal images[END_REF] are examples of systems available to understand the effects of environments on root development. However, ensuring concentrations of nutrients in growth media are within the levels recorded in soil would help to guarantee that root responses observed are physiologically relevant.

HOW TO IMAGE ROOT SYSTEMS?

The 2D agar-based culture of Arabidopsis discussed by Dubrovsky and Forde (2011) is an appealing simple solution to obtain descriptions of the root system at low cost and high throughput. In this respect, aspects of root system phenotypes (root elongation, root angular spread, numbers of root axes, etc.) measured for crop seedlings in simplified 2D systems have been shown to be closely correlated with root properties in 3D and in 2D for mature plants [START_REF] Liao | Effect of phosphorus availability on basal root shallowness in common bean[END_REF][START_REF] Manschadi | The role of root architectural traits in adaptation of wheat to water-limited environments[END_REF][START_REF] Hargreaves | Measuring root traits in barley (Hordeum vulgare ssp vulgare and ssp spontaneum) seedlings using gel chambers, soil sacs and X-ray microtomography[END_REF].

Nevertheless, several other valuable methods have been developed for describing root architecture of crop species in 2D and 3D. These include growth on moistened germination paper rolls or pouches, sand rhizotrons, rhizoboxes, in compost followed by washing, soil columns and gelbased systems where phenotypic effects can be imaged using flatbed scanners, digital cameras, lasers, or even x-ray computed tomography (CT) [START_REF] Hetz | Isolation and characterisation of rtcs, a mutant deficient in the formation of nodal roots[END_REF][START_REF] Whiting | Positive responses to Zn and Cd by roots of the Zn and Cd hyperaccumulator Thlaspi caerulescens[END_REF][START_REF] Bengough | Gel observation chamber for rapid screening of root traits in cereal seedlings[END_REF][START_REF] Fang | 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research[END_REF][START_REF] French | Highthroughput quantification of root growth using a novel image-analysis tool[END_REF][START_REF] Gregory | Root phenomics of crops: Opportunities and challenges[END_REF][START_REF] Hammond | Shoot yield drives phosphorus use efficiency in Brassica oleracea and correlates with root architecture traits[END_REF][START_REF] Iyer-Pascuzzi | Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems[END_REF][START_REF] Trachsel | Shovelomics: High throughput phenotyping of maize (Zea mays L.) root architecture in the field[END_REF][START_REF] Tracy | The X-factor: Visualizing undisturbed root architecture in soils using X-ray computed tomography[END_REF][START_REF] Tracy | Quantifying the effect of soil compaction on three varieties of wheat (Triticum aestivum L.) using x-ray micro computed tomography (CT)[END_REF][START_REF] Chapman | Water supply and not nitrate concentration determines primary root growth in Arabidopsis[END_REF][START_REF] Lobet | A novel image-analysis toolbox enabling quantitative analysis of root system architecture[END_REF][START_REF] Lucas | Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis[END_REF]. Magnetic resonance imaging (for noninvasive analysis of root structures) and positron emission tomography (for analysis of carbon transport and accumulation) can be combined to study the dynamics of structure-function relationships of roots in real soils in a noninvasive manner [START_REF] Jahnke | Combined MRI-PET dissects dynamic changes in plant structures and functions[END_REF]. In all these techniques, a compromise must be made between the disturbance required to allow observations and the resolution and throughput provided by the imaging device. To obtain relevant high-throughput phenotyping data, new (automated) methods and com-binations are still required to provide more natural and nondestructive environmental conditions [START_REF] Nagel | Temperature responses of roots: Impact on growth, root system architecture and implications for phenotyping[END_REF][START_REF] Zhu | From lab to field, new approaches to phenotyping root system architecture[END_REF].

Rice root system architecture can be imaged in gel columns in 3D using optical projection tomography or laser scanning [START_REF] Fang | 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research[END_REF][START_REF] Iyer-Pascuzzi | Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems[END_REF]. For plants grown in soil, x-ray CT techniques and magnetic resonance imaging [START_REF] Heeraman | Three-dimensional imaging of plant roots in situ with X-ray computed tomography[END_REF][START_REF] Nagel | Temperature responses of roots: Impact on growth, root system architecture and implications for phenotyping[END_REF][START_REF] Tracy | The X-factor: Visualizing undisturbed root architecture in soils using X-ray computed tomography[END_REF][START_REF] Lucas | Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis[END_REF][START_REF] Zhu | From lab to field, new approaches to phenotyping root system architecture[END_REF] have recently increased our capabilities to visualize root system architecture in situ nondestructively. For example, the x-ray CT technique can be used to study root architectures under varying nutrient, moisture, temperature, and soil density conditions in a physiologically relevant way over time. Drawbacks of the system, such as imaging time, imaging area, and a 3D image reconstruction approach, are being overcome with improvements in instrumentation (scan time and image quality) and development of new software [START_REF] Mairhofer | RooTrak: Automated recovery of 3D plant root architecture in soil from x-ray micro computed tomography using visual tracking[END_REF][START_REF] Mooney | Marschner Review: Developing X-ray CT to image root architecture in soil[END_REF].

At present, there are various methods for imaging root system architecture as described above, and each approach has its own merit. A transition from 2D to 3D is essential to fully grasp how root system architecture colonizes its environment, but this involves a large increase in expense and data analysis that needs to be taken into account.

ROOT SYSTEM ARCHITECTURE IN THE SOIL

Some biological questions can be sufficiently addressed using agar-based approaches. However, with the importance of the root system contributing to a new green revolution [START_REF] Lynch | Roots of the second green revolution[END_REF][START_REF] Herder | The roots of a new green revolution[END_REF], we should not neglect the key ecological and agronomical aspects.

The study of root architecture has always been compromised by the inherent difficulties in studying a system that necessarily operates in an opaque belowground environment. Some approaches cover Petri dishes with foil, cloth, or wavelength selective filters; the latter allows infrared imaging of root growth [START_REF] Wells | Recovering the dynamics of root growth and development using novel image acquisition and analysis methods[END_REF]. However, the majority of the root phenotyping assays at present are plate based and expose the roots to light, implying artificial conditions in any case. Nevertheless, this has allowed substantial progress over the years. However, depending on the biological question, we need to validate any apparent differences on the level of lateral root density, root angle, etc., by studying root system phenotypes not just in soil, but across a range of varying soil types in terms of texture, structure, and organic matter.

Root architecture is influenced by many soil properties, including mechanical strength and dry bulk density of the soil, the presence and connectivity of air and water-filled pores, soil pH and temperature, and a variety of nutrient and biotic interaction factors that cannot be easily reproduced in highly artificial growth systems. Mechanical impedance in particular is a major physical limitation to root growth that is very difficult to simulate using a gel system, even for the roots of rice [START_REF] Clark | Evaluation of agar and agarose gels for studying mechanical impedance in rice roots[END_REF].

Extracting an entire root system from soil, while maintaining its integrity and avoiding damage to the finer elements of the root system, is an almost impossible challenge. Some promising results have been generated via "shovelomics," a semiquantitative method of excavating, washing, and phenotyping roots, but these have focused mainly on crown root systems [START_REF] Trachsel | Shovelomics: High throughput phenotyping of maize (Zea mays L.) root architecture in the field[END_REF][START_REF] Lynch | Root phenes for enhanced soil exploration and phosphorus acquisition: Tools for future crops[END_REF]. In addition, there are conventional soil coring and underground observation chambers [START_REF] Neill | Comparison of soil coring and ingrowth methods for measuring belowground production[END_REF][START_REF] Lecain | Root biomass of individual species, and root characteristics after five years of CO2 enrichment on native shortgrass steppe[END_REF]. As mentioned above, this can be downscaled using x-ray CT, although the low-throughput and sample size:resolution trade-off remain a constraint [START_REF] Mooney | Marschner Review: Developing X-ray CT to image root architecture in soil[END_REF]. Less extensively, magnetic resonance imaging has been used in a similar way to study root architecture in situ, as has neutron tomography [START_REF] Heeraman | Three-dimensional imaging of plant roots in situ with X-ray computed tomography[END_REF]. In all these approaches, however, cost, effort, the limited throughput, and accessibility (e.g., limited availability of synchrotron beam time for neutron tomography) are still major drawbacks. However, recent technical developments promise increased throughput in the near future.

USING MATHEMATICAL SIMULATION AND MODELING

Despite recent advances in imaging, there are still many processes that cannot be observed experimentally. For these reasons, lateral root research is making increasing use of simulation and mathematical models to understand the way roots function [START_REF] Lynch | Roots of the second green revolution[END_REF][START_REF] Laskowski | Root system architecture from coupling cell shape to auxin transport[END_REF][START_REF] Lucas | Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation[END_REF]. Simple models are useful tools to interpret experiments. Models can test and validate biological hypotheses, and models can be inverted to determine the hidden parameters of a system [START_REF] Dupuy | Root growth models: Towards a new generation of continuous approaches[END_REF]. Models can also integrate complex environmental and developmental variables, including, for example, response to water and nutrient supply [START_REF] Dunbabin | Modelling the interactions between water and nutrient uptake and root growth[END_REF][START_REF] Draye | Model-assisted integration of physiological and environmental constraints affecting the dynamic and spatial patterns of root water uptake from soils[END_REF], various phosphorus concentrations [START_REF] Fang | 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research[END_REF], root adaptation to low nitrogen soil under carbon flux modifications [START_REF] Brun | To what extent may changes in the root system architecture of Arabidopsis thaliana grown under contrasted homogenous nitrogen regimes be explained by changes in carbon supply? A modelling approach[END_REF], and the formation of root cortical aerenchyma in response to soil nutrient status [START_REF] Postma | Root cortical aerenchyma enhances the growth of maize on soils with suboptimal availability of nitrogen, phosphorus, and potassium[END_REF]. Software packages are also available to facilitate the (re)construction of root systems, such as SimRoot [START_REF] Lynch | SimRoot: Modelling and visualization of root systems[END_REF]. Recent progress in elucidating the biological, chemical, and physical processes affecting root growth in soil allows models to be constructed that integrate fundamental regulatory mechanisms into powerful mathematical frameworks incorporating both variability and plasticity [START_REF] De Dorlodot | Root system architecture: opportunities and constraints for genetic improvement of crops[END_REF][START_REF] Draye | Model-assisted integration of physiological and environmental constraints affecting the dynamic and spatial patterns of root water uptake from soils[END_REF]. Taken together, these tools will help understanding the system and knowledge gaps and/or capture and predict the relevant properties of a root system.

CONCLUSION

We agree with Dubrovsky and Forde (2011) that the use of standardized definitions and standard protocols can help to compare data in the scientific community. However, we also think it is important to avoid uniform rules and growth conditions that may be unnecessarily restrictive. To investigate root branching, we should take advantage of the variation that provides unexpected insights. In addition, the value of a screening platform is completely dependent on the trait of interest, and as such, there is no single ideal platform for all root characteristics. Using several approaches to uncover the hidden half of plants is the best way to move forward. These approaches, as described here and by Dubrovsky and Forde (2011), will, when put together, balance out the weaknesses any single approach has.

Address correspondence to Ive.De_Smet@nottingham. ac.uk. www.plantcell.org/cgi/doi/10.1105/tpc.111.094292The Plant Cell, Vol.

24: 15-20, January 2012, www.plantcell.org 2012 American Society of Plant Biologists. All rights reserved.

ACKNOWLEDGMENTS

We thank Jocelyn Malamy for useful suggestions. This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship (BB_BB/H022457/1 to I.D.S.), a Marie Curie European Reintegration Grant (PERG06-GA-2009-256354 to I.D.S.), the Research Foundation Flanders (FWO09/PDO/064 A 4/5 SDS to I.D.S.), Communaute ´franc ¸aise de Belgique (ARC 11/16-036 to X.D.), a European Molecular Biology Organization Long-Term Fellowship (ALTF 503-2010 to B.P.), National Science Foundation ISO-0950866 (M.L.), Deutsche Forschungsgemeinschaft HO2249/8-1 (F.H.), BBSRC BB/G013969/1 (M.R.B. and J.H.), National Science Foundation Plant Genome Research Program grant DBI 0820624 (J.P.L.), U.K. Department for the environment, food and rural affairs project WU0128 (J.H.), European Union FP7 Grant 289300, a Plant KBBE2009 grant named ROOT, a grant from the Agropolis Foundation Montpellier France ("Rhizopolis" Federative Grant) (P.N., L.L., and M.L.), grants from the Interuniversity Attraction Poles Programme (IUAP VI/33), initiated by the Belgian State, Science Policy Office (T.B.). Work at The James Hutton Institute was supported by the Rural and Environment Science and Analytical Services Division of the Scottish Government (2011-2016). Work in the Benfey lab in this area is funded by grants from the National Science Foundation, National Institutes of Health, and Defense Advanced Research Projects Agency. I.C. acknowledges the Comunidad de Extremadura Research Council for a research grant (GR10084/18.F6.03). H.F. is supported by Grants-in-Aid for Scientific Research on Priority Areas (No. 19060006) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. R.H. was supported by a Netherlands Genomics Initiative Horizon grant (050-71-054) from The Netherlands Organization for Scientific Research. B.P. is supported by the Research Foundation Flanders (grant 3G002911), L.L. is supported by the Institut de recherche pour le de ´veloppement and the The Plant Cell COMMENTARY Re ´gion Languedoc-Roussillon (grant "Chercheur d'Avenir"). M.J.B. acknowledges the support of the BBSRC and Engineering and Physical Sciences Research Council funding to the Centre for Plant Integrative Biology, BBSRC Professorial Research Fellowship funding, and Belgian Scientific policy (BELSPO contract BARN).

AUTHOR CONTRIBUTIONS

All authors contributed equally to this article.

Received December 2, 2011;revised December 15, 2011;accepted December 15, 2011;published January 6, 2012.