Lucie Camut

Thomas Regnault

Mathilde Sirlin-Josserand

Lali Sakvarelidze-Achard

Esther Carrera

Julie Zumsteg

Dimitri Heintz

Nathalie Leonhardt

Maria João

Pimenta Lange

Theo Lange

Jean-Michel Davière

Dr Patrick Achard
email: patrick.achard@ibmp-cnrs.unistra.fr

Root-derived GA_12 contributes to temperature-induced shoot growth in Arabidopsis

Plants are able to sense a few degrees rise in temperature, and appropriately adapt their metabolic and growth processes. To this end, plants produce various signaling molecules that act throughout the plant body. Here, we report that root-derived GA 12 , a precursor of the bioactive gibberellins (GA), mediates thermo-responsive shoot growth in Arabidopsis. Our data suggest that root-to-shoot translocation of GA 12 enables a flexible growth response to ambient temperature changes.

In plants, roots and shoots cooperate to ensure adaptive growth rates to fluctuating environmental cues. For example, the elevation of the temperature causes important changes to plant shape, promoting root and hypocotyl elongation, hyponastic growth and flowering [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF][START_REF] Martins | Brassinosteroid signaling-dependent root responses to prolonged elevated ambient temperature[END_REF][START_REF] Kumar | Transcription factor PIF4 controls the thermosensory activation of flowering[END_REF] . Root-derived signals have been reported since long time to modulate shoot morphology, however, the contribution of these signals to temperature-induced shoot growth remains unclear.

Gibberellins (GA) are a class of phytohormones that play important role in diverse developmental processes, including seed germination, plant growth and flowering [START_REF] Davière | Gibberellin signaling in plants[END_REF] . GA promote growth by activating the destabilization of nuclear DELLA growth repressing proteins [START_REF] Davière | Gibberellin signaling in plants[END_REF] . In recent years, GA pathway has been reported to integrate various environmental signals and in response, to optimize plant growth accordingly [START_REF] Achard | Integration of plant responses to environmentally activated phytohormonal signals[END_REF][START_REF] Colebrook | The role of gibberellin signaling in plant responses to abiotic stress[END_REF] . Notably, diverse studies have pointed out a role for GA signaling in temperaturesensing pathways. Whereas warmer temperatures enhance GA biosynthesis gene expression [START_REF] Stavang | Hormonal regulation of temperature-induced growth in Arabidopsis[END_REF][START_REF] Bai | Gibberellin is involved in inhibition of cucumber growth and nitrogen uptake at suboptimal root-zone temperature[END_REF] , lack of DELLA activity accelerates flowering at lower temperatures [START_REF] Kumar | Transcription factor PIF4 controls the thermosensory activation of flowering[END_REF] . Thus, changes in GA synthesis and/or distribution have important effect on plant growth adaption to fluctuating temperatures. If it is admitted that GA are usually synthetized close to their site of action, they can also be transported throughout the plant [START_REF] Regnault | The gibberellin precursor GA 12 acts as a long-distance growth signal in Arabidopsis[END_REF][START_REF] Binenbaum | Gibberellin localization and transport in plants[END_REF] . This latest observation prompted us to investigate whether root-to-shoot translocation of GA contributes to accelerated shoot growth at elevated ambient temperatures.

To investigate this possibility, we measured the aerial growth of reciprocal grafts between Arabidopsis wild-type (Col-0) and GA-deficient mutant (ga1-3) at 20° and 28°C (Supplementary Figure 1a-c).

Strikingly, whereas the rosettes of Col-0 self-grafts grown at 28°C were larger to those grown at 20°C, the rosette radius of wild-type scions grafted onto ga1-3 rootstocks (ga1-3/Col-0 grafts) was smaller at 28°C (Figure 1a and Supplementary Figure 1b). Moreover, the diameter of three-week-old ga1-3 rosettes grafted onto wild-type rootstocks (Col-0/ga1-3 grafts) was significantly larger at 28°C than at 20°C (Figure 1b and Supplementary Figure 1c). Thus, root-derived GA contribute substantially to the shoot growth promotion triggered by high temperature. Remarkably, we found that ga20ox1-2-3 triple mutant rootstocks, which produce GA 12 but are unable to convert it into GA 15 and following products [START_REF] Hedden | Gibberellin biosynthesis and its regulation[END_REF] (Supplementary Figure 1a), were also able to increase the growth of ga1-3 grafted scions at high temperature (Figure 1a,b and Supplementary Figure 1b,c); consistent with the premise that GA 12 is a mobile growth signal in Arabidopsis 9 .

To determine the nature of the root-derived GA signal leading to accelerated shoot growth at high temperature, we quantified the GA levels in scions of Col-0/ga1-3 and ga1-3/Col-0 grafts, and wildtype and ga1-3 self-graft controls. Consistent with the morphological parameters, bioactive GA 4 accumulated to higher levels in ga1-3 mutant scions of Col-0/ga1-3 grafts growing at 28°C compared with those at 20°C (Figure 1c). Interestingly, GA 12 was also detected in higher amount in ga1-3 mutant scions of Col-0/ga1-3 grafts grown at 28°C. Thus, these data indicate that high temperatures increase the level of root-borne GA 12 delivered to the shoots, which is then converted into GA 4 by the activities of GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox) [START_REF] Hedden | Gibberellin biosynthesis and its regulation[END_REF] (Supplementary Figure 1a).

Consistent with this hypothesis, we found that GA 4 accumulated to higher level in wild-type scions of Col-0/Col-0 grafts compared with ga1-3/Col-0 grafts at 28°C (Figure 1c), and therefore could explain their difference in rosette diameter (Figure 1a). Unlike GA 4 , GA 12 was detected at a lower level in wild-type scions of Col-0/Col-0 and ga1-3/Col-0 grafts grown at 28°C compared with those grown at 20°C (Figure 1c). In wild-type shoots, the concentration of GA 12 is determined by both the amount of root-derived and shoot-synthetized GA 12 , and the rate of its metabolic conversion to GA 15 by the activity of GA20ox, which catalyze a rate-limiting step in GA metabolism [START_REF] Hedden | Gibberellin biosynthesis and its regulation[END_REF] . As previously reported [START_REF] Stavang | Hormonal regulation of temperature-induced growth in Arabidopsis[END_REF] , we found that elevated ambient temperature enhanced GA20ox1 expression, and thereby, the conversion of GA 12 into GA 15 (Figure S1d and Figure 1c). Thus, warm temperatures enhance both GA 12 supply and metabolic conversion in wild-type shoots.

High ambient temperature could enhance the delivery of root-borne GA 12 in the shoots via increased synthesis of GA 12 in roots and/or enhanced transport of GA 12 from the roots. To examine these possibilities, we first determined the concentration of endogenous GA in wild-type roots grown at 20° and at 28°C. The content in GA 12 was not statistically different between the two conditions (Figure 1d), as well as the expression of GA metabolism genes catalyzing the first steps of the GA biosynthetic pathway (Supplementary Figure 1e). We also determined the concentration of GA 12 in xylem exudates collected from shoots of five-week-old Col-0/ga1-3 grafts grown at 20° and 28°C.

Remarkably, both the concentration of GA 12 and the GA 12 /K + ratio (K + is used as a reference of the xylem sap flux [START_REF] Gaymard | Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap[END_REF] , which fluctuates with the transpiration rate of the leaves) were increased at 28°C (Figure 1e). Altogether, these findings provide compelling evidence that high temperature increases the root-to-shoot translocation of GA 12 , which in turn, after metabolic conversion into bioactive GA 4 , promotes shoot elongation.

In Arabidopsis, thermo-sensing pathway is dependent on the transcription factor PIF4, which accelerates shoot growth through direct activation of auxin biosynthesis and signaling genes [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF][START_REF] Kumar | Transcription factor PIF4 controls the thermosensory activation of flowering[END_REF] . It has been reported that warm temperature enhances both PIF4 expression and transcriptional activity, while GA-regulated DELLA proteins repress PIF4 activity by preventing its DNA binding capacity (Figure 2a) [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF][START_REF] Kumar | Transcription factor PIF4 controls the thermosensory activation of flowering[END_REF][START_REF] De Lucas | A molecular framework for light and gibberellin control of cell elongation[END_REF] . To investigate whether root-derived GA 12 has an effect on PIF4 activity in shoots, we generated a transgenic line that produces GA 12 solely in the root. To this end, we complemented the kao1 kao2 double mutant (deprived of GA 12 synthesis [START_REF] Regnault | The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development[END_REF] ) with a construct that expresses KAO1 only in root, with the use of the ROOT-SPECIFIC KINASE 1 (ARSK1) promoter (in kao1 kao2 pARSK1:KAO1-RFP). Indeed, as previously observed with pARSK1:GUS reporter line [START_REF] Hwang | An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl[END_REF] , the promoter of ARSK1 is active in the entire root (except the root apex), mainly in epidermal and endodermal cells, but not in the hypocotyl and cotyledons (Supplementary Figure 2a-e). Moreover, to further validate the root-specific activity of ARSK1 promoter, we also expressed GA20ox1 under the regulation of the ARSK1 promoter in the ga20ox1-2-3 triple mutant (in ga20ox1-2-3 pARSK1:GA20ox1-RFP).

Consistent with the assumption that GA 12 is the main GA form translocated from root to shoot, pARSK1:KAO1-RFP entirely rescued the dwarf phenotype of kao1 kao2 mutant seedlings (in kao1 kao2 pARSK1:KAO1-RFP) grown at 22°C in long day conditions, while pARSK1:GA20ox1-RFP only restored the growth of ga20ox1-2-3 roots (in ga20ox1-2-3 pARSK1:GA20ox1-RFP); the hypocotyls being similar from that of ga20ox1-2-3 triple mutant (Supplementary Figure 3a,b). Thus, the activity of the ARSK1 promoter is restricted to the root at seedling stage. At adult stage, ARSK1 promoter is also active in rosette leaves (but not in inflorescence; Supplementary Figure 3e-i), and therefore, all further experiments were performed on 7-d-old seedlings. Noteworthy, complementary analysis revealed that ambient temperature does not modulate ARSK1 promoter activity (Supplementary Figure 2f,g).

To further substantiate the previous results obtained with the grafts (Figure 1), we finally examined the hypocotyl length of kao1 kao2 pARSK1:KAO1-RFP and ga20ox1-2-3 pARSK1:GA20ox1-RFP seedlings at 20° and 28°C, compared to that of wild-type and pif4-101 mutant. For this experiment, the seedlings were grown in short day conditions under low light intensity (30 µmol/m 2 /s) to enhance hypocotyl elongation and therefore to monitor subtle changes in hypocotyl length. Whereas the seedlings exhibited similar growth patterns at 20°C (moderate temperature inhibits PIF action 1 ), the hypocotyls of wild-type and kao1 kao2 pARSK1:KAO1-RFP were more elongated than those of ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 mutant at 28°C (Figure 2b,c). Consistent with this result, we found that hypocotyl growth of rootless wild-type seedlings (hence producing GA only in shoot) was significantly reduced at 28°C compared to that of intact wild-type seedlings (producing GA in both root and shoot), but less affected than pif4 mutant, which is substantially resistant to temperature-induced shoot growth 3 (Supplementary Figure 4a). Furthermore, we found that elevated temperature decreased the abundance of the DELLA protein REPRESSOR OF GA1-3 (RGA) in hypocotyls of wild-type, kao1 kao2 pARSK1:KAO1-RFP and pif4-101 mutant (compared to that at 20°C) but not in ga20ox1-2-3 pARSK1:GA20ox1-RFP hypocotyls (Figure 2d and Supplementary Figure 4b); hence confirming that GA signaling acts upstream of PIF4. Thus, increased root-to-shoot translocation of GA 12 at 28°C enhances DELLA protein degradation, and as a consequence shoot growth, in accord with the growth parameters obtained with the grafts in Figure 1.

Subsequently, we analyzed the effects of root-to-shoot translocation of GA 12 on the expression of PIF4 target genes in hypocotyls after a temperature shift from 20° to 28°C for 4h. Consistent with the above results, we found that INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), IAA29, YUCCA 8 (YUC8) and PACLOBUTRAZOL RESISTANT 1 (PRE1) transcripts were increased by about twofold in wildtype and kao1 kao2 pARSK1:KAO1-RFP hypocotyls treated at 28°C, compared with those kept at 20°C (Figure 2e). By contrast, high temperature did not significantly induce the expression of the PIF4 target genes in ga20ox1-2-3 pARSK1:GA20ox1-RFP hypocotyls. Given that DELLA-PIF4 interaction blocks the DNA binding capacity of PIF4 to its target genes [START_REF] De Lucas | A molecular framework for light and gibberellin control of cell elongation[END_REF] , these results suggest that root-derived GA 12 induces PIF4-mediated temperature-responsive gene expression, by enhancing the destabilization of DELLA proteins.

Plant hormones play a critical role in long-distance communication, ensuring coordinated developmental processes in response to fluctuating environmental cues. For example, nitrate availability fine-tunes the amount of root-borne cytokinins delivered to the shoots to optimize their development [START_REF] Osugi | Systemic transport of trans-zeatin and its precursor have differing roles in Arabidopsis shoots[END_REF] . Remarkably, whereas previous studies have revealed that GA can move over long distances [START_REF] Regnault | The gibberellin precursor GA 12 acts as a long-distance growth signal in Arabidopsis[END_REF][START_REF] Binenbaum | Gibberellin localization and transport in plants[END_REF] , the physiological relevance of this transport remained unclear. In this work, we demonstrated that root-derived GA 12 contributes substantially to temperature-induced shoot growth, consistent with previous data showing that GA 12 is the main mobile GA signal over long distances in Arabidopsis [START_REF] Regnault | The gibberellin precursor GA 12 acts as a long-distance growth signal in Arabidopsis[END_REF] . The amount of root-borne GA 12 delivered to the shoots depends on its concentration in xylem sap and the leaf transpiration, which increases at elevated temperature. By analyzing the GA 12 /K + ratio, our findings have revealed a specific effect of the temperature on the concentration of GA 12 in xylem fluid (Figure 1e). Although further studies are needed to unravel the molecular mechanism, it is tempting to speculate that ambient temperature regulates the activity or the level of GA efflux transporters facilitating the translocation of GA 12 into the xylem. Unfortunately, despite the recent identification of several GA influx transporters involved in the local movement of GA in Arabidopsis [START_REF] Binenbaum | Gibberellin localization and transport in plants[END_REF][START_REF] Saito | The jasmonate-responsive GTR1 transporter is required for gibberellin-mediated stamen development in Arabidopsis[END_REF][START_REF] Tal | The Arabidopsis NPF3 protein is a GA transporter[END_REF] , GA efflux transporters remain to be discovered.

Climate change has begun to exert significant effects in plant morphology and behavior. Pioneering studies suggested that environmental signals such as flooding or soil temperature influence shoot growth, at least in part, via the modulation in the supply of GA in the xylem sap in tomato and in pine [START_REF] Reid | The effects of flooding on the export of gibberellins from the root to the shoot[END_REF][START_REF] Lavender | Spring shoot growth in Douglas-fir may be initiated by gibberellins exported from the roots[END_REF] . Although it remains unclear how the temperature signal is sensed in the root, our results show that root-derived GA 12 permits a flexible growth response to ambient temperature changes (Supplementary Figure 5; Supplementary Discussion). Root-to-shoot translocation of GA 12 might play an essential role in response to day and night air temperature oscillations for which, in contrast to soil temperature, the amplitude of the variations can rapidly rise in spring.

Methods

Plant material and growth conditions

Mutant lines are derived from Columbia-0 (Col-0) (ga1-3; kao1-1 kao2-1; ga20ox1 ga20ox2 ga20ox3-1; pif4-101) backgrounds. Plants were grown on soil or on plates containing 1x Murashige-Skoog (MS) medium (Duchefa Biochemical), 1% sucrose and 0.8% agar, at 20°C or at 28°C, as indicated.

GA-deficient mutant seeds were pretreated at 4°C with 5 µM GA 3 (Sigma-Aldrich) for 3 days to synchronize germination, washed thoroughly 3 times, then surface sterilized before sowing.

Plasmid construction and plant transformation

To generate pARSK1:GUS, pARSK1 promoter (2.7-kb fragment) was PCR amplified from Col-0 genomic DNA with appropriate primers (listed in Supplementary Table 3) and inserted into

Grafting

Micrografting between hypocotyls of rootstocks and scions was carried out without collars on 6-dayold seedlings, as previously described [START_REF] Turnbull | Micrografting techniques for testing long-distance signalling in Arabidopsis[END_REF] . Successful grafts were transferred into soil and grown under continuous light at 20°C or at 28°C.

Xylem sap exudations

Stems of 5-week-old grafts (5 days after bolting) were decapitated 2 cm above the rosette with a razor blade and placed in closed box to maintain high humidity. Exudate was collected every 30 min for 3 h (the first drop was discarded), pooled and concentrated under vacuum centrifugation (Savant ThermoFisher).

GA determinations

GA contents in scions of grafted plants (Figure 1c) were determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) using a Q-Exactive spectrometer (Orbitrap detector; ThermoFisher Scientific). Dry grounded material was suspended in 80% methanol-1% acetic acid including 17-2 H 2 -labeled GA internal standards (Olchemim) and mixed by shaking during one hour at 4ºC. The extract was kept at -20ºC overnight, centrifuged and the supernatant dried in a vacuum evaporator. The dry residue was dissolved in 1% acetic acid and passed consecutively through a reverse phase column Oasis HLB column and a cationic exchange Oasis MCX eluted with MeOH.

The dried eluate was dissolved in 5% acetonitrile-1% acetic acid, and the GAs were separated by UHPLC (Accucore C18 column 2.6 µm, 100 mm x 2.1 mm; ThermoFisher Scientific) with a 2 to 55% acetonitrile gradient containing 0.05% acetic acid, at 400 µL/min over 21 min. The concentrations of GAs in the extracts were analyzed by selected ion monitoring (SIM) using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1programs. The total dry masses of the samples are given in Supplementary Table 4.

Quantitative analysis of endogenous GA levels in roots (Figure 1d) was performed as described previously [START_REF] Lange | Gibberellin biosynthesis in developing pumpkin seedlings[END_REF] , with the following modifications. After solvent partitioning the samples were directly loaded onto C18 cartridges (Waters), and GA 51 and GA 34 were eluted with 50%, GA 9 , GA 24 , GA 4 and GA 15 with 60%, and GA 12 with 70% methanol-water, pH 3 (acetic acid). The derivatized samples were analysed by gas chromatography-mass spectrometry (GC-MS) as described elsewhere [START_REF] Lange | Gibberellin biosynthesis in developing pumpkin seedlings[END_REF] .

GA 12 content in xylem sap collected from ga1-3 scions of Col-0/ga1-3 grafted plants (Figure 1e) was determined by ultra-performance liquid chromatography-tandem mass spectrometry (Thermo Scientific Dionex UltiMate 3000 UHPLC coupled MS Bruker EVOQ Elite) as described previously [START_REF] Regnault | The gibberellin precursor GA 12 acts as a long-distance growth signal in Arabidopsis[END_REF] .

K + determination

Xylem sap exudation samples were mineralized in concentrated HNO 3 90 min at 160 °C. The potassium concentration in the solution was determined using inductively coupled plasma optical emission spectroscopy (ICP AES Vista MPX).

Gene expression analyses

Total RNA from hypocotyls, roots and grafted shoots were extracted with NucleoSpin RNA Plant kit (Macherey Nagel). RNA samples were treated with DNase I (Promega) and reverse transcribed into complementary DNA (cDNA) with Superscript IV reverse transcriptase (Invitrogen). The cDNA was further quantified with a SYBR Green Master mix (Roche) and gene-specific primers (listed in Supplementary Table 3) on a Lightcycler LC480 apparatus (Roche) as previously described [START_REF] Regnault | The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development[END_REF] .

AT4G34270 (TIP41-LIKE) and AT4G26410 genes were used as internal reference genes. Data are means ± s.d. of three biological samples.

Protein gel blot analyses

Protein extraction and immunoblot analyses were as previously described [START_REF] Regnault | The gibberellin biosynthetic genes AtKAO1 and AtKAO2 have overlapping roles throughout Arabidopsis development[END_REF] . Total proteins from hypocotyls of 7-d-old seedlings were extracted in 2x SDS-PAGE buffer and fractionated on a 10% SDS-PAGE gel. Immunoblots were performed using a 2000-fold dilution of anti-RGA antibody (Agrisera, product AS11 1630, lot 1511). The blot was subsequently probed with anti-cdc2 (PSTAIRE) antibody (Santa Cruz Biotechnology) for loading control. Quantification of the signals was performed using ImageJ package version 1.48v (www.imageJ.nih.gov). Similar results were obtained in two independent experiments.

GUS analyses

pARSK1:GUS seedlings were fixed for 10 min in 90% (v/v) acetone on ice, washed, then infiltrated in GUS solution (500 µg/ml 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Gluc); 50 mM sodium phosphate pH 7; 1 mM potassium ferricyanide; 1 mM potassium ferrocyanide; 10 mM EDTA; 0.01% triton X-100) for 15 min and incubated at 37°C for 8 h. Then, the GUS solution was replaced with 100% (v/v) ethanol during 6 h at room temperature and kept in 70% (v/v) ethanol at 4°C.

Statistical analysis

Statistical analyses were performed using RSudio package version 1.2.1335 (www.rstudio.com).

Phenotypic characterization and qRT-PCR experiments were analyzed using Student's t-test (P < 0.05) or by analysis of variance (ANOVA). Tukey's Honest Significant Difference (HSD) was used to compare between genotypes using a significance threshold of 5%. T-test and ANOVA analyses can be found in Supplemental Dataset 1. The exact total number of independent rosette (n) for each graft combination is listed in Supplementary Table 1. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. Asterisks indicate that plants had already initiated flowering before measuring their rosette diameter. c, Concentrations of GAs (ng.g -1 dry weight ± s.d.) in the shoots of 12 days post-grafted plants of indicated genotypes, grown at 20°C (highlighted in blue) or 28°C (highlighted in red). Notation of genotype is rootstock/scion. In samples marked n.d. endogenous GA was not detected, but internal standard was recovered. The values are means of three biological replicates except where indicated. # , Two biological replicates only. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. d, Concentration of GAs (ng.g - 1 dry weight ± s.d.) in the roots of Col-0 seedlings grown for 8 days at 20°C (highlighted in blue) or for 6 days at 20°C and 2 days at 28°C (highlighted in red). Values are means of three biological replicates. There is no significant differences (p < 0.05) for 28°C versus 20°C by two-way Student's ttest. The exact p value for each comparison can be found in Supplementary Dataset 1. e, GA 12 content (nM ± s.d.), K + content (mM ± s.d.) and GA 12 /K + ratio (10 -6 ± s.d.) in xylem exudates of Col-0/ga1-3 grafted plants grown at 20°C for 5 weeks (highlighted in blue) or for 4 weeks at 20°C and 1 week at 28°C (highlighted in red). Notation of genotype is rootstock/scion. Values are means of three biological replicates. Asterisks indicate significant differences (p < 0.05) for 28°C versus 20°C by two-way Student's t-test. The exact p value for each comparison can be found in Supplementary Dataset 1. , in SD conditions. The exact total number of independent seedling (n) for each genotype is listed in Supplementary Table 2. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. d, Immunodetection of RGA protein in hypocotyls of 7-d-old Col-0, kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown at 20°C or 28°C, in SD conditions. PSTAIRE serves as sample loading control. Values indicate RGA signal intensity relative to PSTAIRE signal intensity. Similar results were obtained in two independent experiments (shown in Supplementary Figure 4b). e, Expression levels (mean) of IAA19, IAA29, YUC8 and PRE1 in hypocotyls of wild-type (Col-0), kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown in short-day conditions (30 µmol/m 2 /s) at 20°C (in blue) or at 20°C and then transferred at 28°C for 4 h under light (in red). Error bars indicate s.d. (n = 3 biologically independent experiments). Different letters denote significant differences (p < 0.01) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1.

  pDONR207 (Invitrogen) by Gateway cloning and recombined with pGWB633 21 . To generate pARSK1:KAO1-RFP and pARSK1:GA20ox1-RFP constructs, pARSK1 (2.7-kb fragment) was cloned into HindIII/SpeI sites of pB7RWG2 22 , replacing the p35S promoter. KAO1 and GA20ox1 cDNA amplified by RT-PCR were then inserted into pDONR207 and recombined with the new pARSK1-pB7RWG2 vectors. The plant binary vectors were introduced into Agrobacterium tumefaciens GV3101 strain by electroporation, and Arabidopsis Col-0, kao1 kao2 and ga20ox1-2-3 plants were transformed by floral dip to respectively obtained pARSK1:GUS, kao1 kao2 pARSK1:KAO1-RFP and ga20ox1-2-3 pARSK1:GA20ox1-RFP transgenic lines.

Figure 1 .

 1 Figure 1. Root-derived GA are essential for the thermal induction of shoot growth. a-b, Feret's rosette diameter (mean ± s.d.) of Col-0/Col-0, ga1-3/Col-0, ga1-3/ga1-3, Col-0/ga1-3 and ga20ox1-2-3/ga1-3 grafts grown at 20°C (in blue) or 28°C (in red), two-(a) and three-weeks (b) post-grafting.The exact total number of independent rosette (n) for each graft combination is listed in Supplementary Table1. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. Asterisks indicate that plants had already initiated flowering before measuring their rosette diameter. c, Concentrations of GAs (ng.g -1 dry weight ± s.d.) in the shoots of 12 days post-grafted plants of indicated genotypes, grown at 20°C (highlighted in blue) or 28°C (highlighted in red). Notation of genotype is rootstock/scion. In samples marked n.d. endogenous GA was not detected, but internal standard was recovered. The values are means of three biological replicates except where indicated. # , Two biological replicates only. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. d, Concentration of GAs (ng.g - 1 dry weight ± s.d.) in the roots of Col-0 seedlings grown for 8 days at 20°C (highlighted in blue) or for 6 days at 20°C and 2 days at 28°C (highlighted in red). Values are means of three biological replicates. There is no significant differences (p < 0.05) for 28°C versus 20°C by two-way Student's ttest. The exact p value for each comparison can be found in Supplementary Dataset 1. e, GA 12 content (nM ± s.d.), K + content (mM ± s.d.) and GA 12 /K + ratio (10 -6 ± s.d.) in xylem exudates of Col-0/ga1-3 grafted plants grown at 20°C for 5 weeks (highlighted in blue) or for 4 weeks at 20°C and 1 week at 28°C (highlighted in red). Notation of genotype is rootstock/scion. Values are means of three biological replicates. Asterisks indicate significant differences (p < 0.05) for 28°C versus 20°C by two-way Student's t-test. The exact p value for each comparison can be found in Supplementary Dataset 1.

Figure 2 .

 2 Figure2. Root-borne GA 12 enhances hypocotyl elongation at 28°C via a DELLA-dependent mechanism. a, Schematic of the GA and thermo-responsive growth regulation. b, Representative 7day-old wild-type (Col-0), kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown in short day (SD) conditions (30 µmol/m 2 /s) at 20°C or 28°C. Scale bars represent 5 mm. c, Hypocotyl length (mean ± s.d.) of 7-day-old Col-0, kao1 kao2, kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown at 20°C (in blue) or 28°C (in red), in SD conditions. The exact total number of independent seedling (n) for each genotype is listed in Supplementary Table2. Different letters denote significant differences (p < 0.05) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1. d, Immunodetection of RGA protein in hypocotyls of 7-d-old Col-0, kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown at 20°C or 28°C, in SD conditions. PSTAIRE serves as sample loading control. Values indicate RGA signal intensity relative to PSTAIRE signal intensity. Similar results were obtained in two independent experiments (shown in Supplementary Figure4b). e, Expression levels (mean) of IAA19, IAA29, YUC8 and PRE1 in hypocotyls of wild-type (Col-0), kao1 kao2 pARSK1:KAO1-RFP, ga20ox1-2-3 pARSK1:GA20ox1-RFP and pif4-101 seedlings grown in short-day conditions (30 µmol/m 2 /s) at 20°C (in blue) or at 20°C and then transferred at 28°C for 4 h under light (in red). Error bars indicate s.d. (n = 3 biologically independent experiments). Different letters denote significant differences (p < 0.01) using one-way ANOVA with Tukey's test for multiple comparisons. The exact p value for each comparison can be found in Supplementary Dataset 1.

Acknowledgements

We thank Tp. Sun for providing seeds of ga1-3 (Col-0 background), P. Hedden for providing ga20ox1-2-3. This work was supported by the Centre National de la Recherche Scientifique and the French ministry of research and higher education.

Data availability

All data generated or analyzed during this study are included in the published article and its Supplementary information.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Author contributions

L.C., T.R., L.S.A., E.C., J.Z., D.H., N.L., M.J.P.L, T.L., J.M.D. and P.A. performed experimental work; L.C., T.R., D.H., N.L., M.J.P.L., T.L., J.M.D. and P.A. designed the experiments; M.S., M.J.P.L., T.L., J.M.D. and P.A. realized the figures and wrote the paper.

Correspondence and requests for materials should be addressed to P.A.

Competing interests

The authors declare no competing interests.

Lucie Camut [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF][START_REF] Martins | Brassinosteroid signaling-dependent root responses to prolonged elevated ambient temperature[END_REF] , Thomas Regnault [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF][START_REF] Martins | Brassinosteroid signaling-dependent root responses to prolonged elevated ambient temperature[END_REF][START_REF] Kumar | Transcription factor PIF4 controls the thermosensory activation of flowering[END_REF] , Mathilde Sirlin-Josserand 1 , Lali Sakvarelidze-Achard [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF] , Esther Carrera [START_REF] Davière | Gibberellin signaling in plants[END_REF] , Julie Zumsteg 1 , Dimitri Heintz 1 , Nathalie Leonhardt 5 , Maria João Pimenta Lange [START_REF] Colebrook | The role of gibberellin signaling in plant responses to abiotic stress[END_REF] , Theo Lange [START_REF] Colebrook | The role of gibberellin signaling in plant responses to abiotic stress[END_REF] , Jean-Michel Davière [START_REF] Quint | Molecular and genetic control of plant thermomorphogenesis[END_REF] and Patrick Achard 1 *

Supplementary Discussion

Here, we develop few points that were only shortly discussed in the Brief Communication article. In our work, we have shown by using grafting (Col-0/ga1-3 graft combination; Fig. 1) and transgenic plants (kao1 kao2 pARSK1:KAO1-RFP; Fig. 2), which both produce GA solely in the root, that rootderived GA 12 contributes substantially to temperature-induced shoot growth. We may regret that these experiments were performed at different developmental stages (at adult and seedling stage, respectively), imposed by the leakiness of the ARSK1 promoter activity in rosette leaves (Fig. S3).

Nevertheless, the results obtained with the grafts and the transgenic seedlings clearly support the conclusion that root-borne GA 12 mediates thermo-responsive shoot growth. Another issue is that there is no straightforward experiment to evaluate the physiological relevance of the root-to-shoot transport of GA 12 at elevated temperature, in wild-type plants. The concentration of GA 12 in xylem sap (Fig. 1e) was determined in exudates collected from scions of Col-0/ga1-3 grafts and not from wild-type plants to ensure that GA 12 only derives from root, and not the combination of both root-derived and shootsynthetized GA 12 . Although we cannot exclude that lack of GA-synthesis in ga1-3 scions (in Col-0/ga1-3 grafts) might influence the transport of GA 12 from source (Col-0 root) to sink (ga1-3 shoot) organs, we found that wild-type rosettes of Col-0 self-grafts grown at 28°C were larger than those grafted onto ga1-3 rootstocks (Fig. 1a). Consistent with this result, the hypocotyl length of rootless wild-type seedlings (even though they produce GA in their hypocotyl) was smaller than that of intact wild-type seedlings at 28°C (Fig. S4a). Hence, these results indicate that root-borne GA 12 contributes substantially to enhanced shoot growth at elevated ambient temperature, in Arabidopsis. This adaptive mechanism may have essential role in the development of field grown plants exposed to daily ambient temperature fluctuations. Supplementary Figure 5. Root-derived GA 12 is required for temperature-induced shoot growth. At 20°C, the growth of root and shoot is regulated by GA 4 in a root-and shoot-autonomous manner. At 28°C, warm temperature activates root-to-shoot transport of GA 12 , whose action combined with shoot-synthetized GA 12 , promotes shoot growth, after metabolic conversion into GA 4 . KA, entkaurenoic acid; KAO, ent-kaurenoic acid oxidase; GA20ox, GA 20-oxidase; GA3ox, GA 3-oxidase.

Supplementary Figures


Jérémie Gouyon

Craig Simon

Sophie Griveau

Catherine Sella

Laurent Thouin

Fethi Bedioui

Anne Varenne
email: anne.varenne@chimieparistech.psl.eu

Fanny D'orlyé

Reversible microfluidics device for precious metal electrodeposition and depletion yield studies

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Printed circuit boards (PCBs) are known as an integral part of our technological devices (smartphones, computers…). As their use has become more and more important in the recent decades, the amount of waste is increasing and becomes an ecological, economic and strategic challenge [1,[START_REF] Goodship | Waste Electrical and Electronic Equipment (WEEE) Handbook[END_REF]. Indeed, waste PCBs are a major source of strategic metals [START_REF] Ghosh | Waste Printed Circuit Boards recycling: an extensive assessment of current status[END_REF], such as platinum group metals (PGM), rare earth elements or other expensive species. Their recovery is crucial, especially since some of them are defined as critical (supply risks, high cost) by the European Commission [START_REF] Of | Report on critical raw materials and the circular economy[END_REF] since 2014, such as Platinum (Pt) or Palladium (Pd). Their recycling is generally carried out in a three steps process [START_REF] Kaya | Recovery of metals and nonmetals from electronic waste by physical and chemical recycling processes[END_REF]: (i) a mechanical grinding to reduce PCBs to powders and separate ferrous and non-ferrous metals, (ii) pyrolysis at 1000°C to remove the organic constituents and (iii) a hydrometallurgical treatment to selectively remove metals in ionic form in leaching solutions. These treatments are thus promising in terms of strategic metals recovery rate, but purification methods are still time-consuming and need to be optimized to ensure high purity of recycled materials, especially in their metallic form.

Thanks to microfluidics [START_REF] Whitesides | The origins and the future of microfluidics[END_REF], the change in scale from an industrial process to microchannel results in a modification of the mechanical properties of fluids. This allows leading to high conversion for organic synthesis, for example, and high purity of the products [START_REF]Les micro-réacteurs : opportunités et applications pour les industries chimiques -2007[END_REF][START_REF] Suryawanshi | A review on microreactors: Reactor fabrication, design, and cutting-edge applications[END_REF]. In addition, the low manufacturing cost of microreactors permits them to be parallelized, allowing for processing a large quantity of samples, thus competing with current industrial macro-processes [START_REF] Roberge | Industrial design, scale-up, and use of microreactors[END_REF]. Even if diagnosis is one of the main application of microfluidics (for environmental [START_REF] Yew | A Review of State-of-the-Art Microfluidic Technologies for Environmental Applications: Detection and Remediation[END_REF] and biological [START_REF] Tian | Recent advances in microfluidic technologies for organon-a-chip[END_REF] survey), the use of such miniaturized tools for the treatment of large amounts of liquid is also feasible.

Microfluidic coupled to the integration of electrodes within microchannels has been previously used to remove potential interfering agents from real samples but also to increase the selectivity towards a target (such as oxalic acid or nitric oxide) during its electrochemical detection [START_REF] Watanabe | Fabrication of a Microfluidic Device with Boron-doped Diamond Electrodes for Electrochemical Analysis[END_REF][START_REF] Oliveira | Development of a flow microsensor for selective detection of nitric oxide in the presence of hydrogen peroxide[END_REF].

The developed setups are composed of a four-electrodes cell, where two working electrodes (WE) are placed perpendicularly to a single microchannel containing the solution under continuous flow. Watanabe et al. [START_REF] Watanabe | Fabrication of a Microfluidic Device with Boron-doped Diamond Electrodes for Electrochemical Analysis[END_REF] used a large WE (WE DEP , depletion electrode) in order to remove the electroactive analyte (ferricyanide) from the solution and checked the depletion yield by measuring ferricyanide concentration thanks to the second WE (WE det , detection electrode) positioned downstream. The importance of main parameters on the depletion yield has been discussed by the authors such as ratio of electrode width to channel height, flow velocity and applied potentials. The authors stated that their setup was not suitable to complete the depletion up to 100 % due to the height of the channel which was larger than the convection-diffusion layer of the sample. Indeed, the thin-layer regime at the electrode must be achieved under mass transport control for complete depletion [START_REF] Amatore | Theory and Experiments of Transport at Channel Microband Electrodes under Laminar Flows. 1. Steady-State Regimes at a Single Electrode[END_REF][START_REF] Amatore | Theory and Experiments of Transport at Channel Microband Electrodes under Laminar Flows. 2. Electrochemical Regimes at Double Microband Assemblies under Steady State[END_REF][START_REF] Oliveira | Direct Electroanalytical Method for Alternative Assessment of Global Antioxidant Capacity Using Microchannel Electrodes[END_REF]. More recently, similar strategy was adopted for the assessment of global antioxidant capacity and for the depletion of hydrogen peroxide (up to 95%) [START_REF] Oliveira | Development of a flow microsensor for selective detection of nitric oxide in the presence of hydrogen peroxide[END_REF]. In this last case, the selectivity of nitric oxide detection was enhanced by positioning a NO-sensor downstream of the depletion electrode. It should be noted that the devices described in the literature for this type of application are sealed and do not offer any possibility of easy and quick reuse in case of pollution of the depletion electrodes and their regeneration.

Reversibly bonded chips were reported in the literature and they were generally used for other applications such as coupling of electrophoresis with electrochemical detection or surface patterning for bio-molecule analysis [START_REF] Anwar | Reversible sealing techniques for microdevice applications[END_REF]. Several strategies for the reversible bonding of the different parts of the microdevices were used such as magnetism, vaccum or spontaneous conformal contact between PDMS and glass [START_REF] Martin | Dual-Electrode Electrochemical Detection for Poly(dimethylsiloxane)-Fabricated Capillary Electrophoresis Microchips[END_REF] or other materials (metals, acrylate plate [START_REF] Petroni | Simple approach for the fabrication of screenprinted carbon-based electrode for amperometric detection on microchip electrophoresis[END_REF]).

They offer the main advantage of facilitating the washing and the renewability of channels. We propose in this study an original microfluidic device to address the issue of recovering Au and Pd dissolved in acidic media (as AuCl 4

-and PdCl 4 2-respectively), mimicking leach liquors from the waste PCBs recycling process. For this purpose, a re-usable new reversible Glass-NOA ® -PDMS microchip with four graphite microbands electrodes adapted from previously developed one by our group [START_REF] Gouyon | Characterization of home-made graphite/PDMS microband electrodes for amperometric detection in an original reusable glass-NOA®-PDMS electrophoretic microdevice[END_REF], was designed to set-up an original amperometric recovery device for both depletion and yield quantification. The device reported here offers the unique advantage of being a micro sandwich system with a reversible bond composed of two parts without the need for a magnetic or other device to maintain perfect adhesion, allowing easy washing of the microchannel and renewal of the electrode surface by simply repositioning the microband electrodes. This configuration permits enhancing its lifetime. First, to optimize the performance of the proposed microdevice, ferrocene methanol was used as a model. Then, the method efficiency for precious metallic species were discussed and compared to theoretical predictions.

Materials and methods

Solutions

The artificial leach liquors were prepared by dilution of appropriate amounts of metallic standard solutions with HCl in ultrapure water so as to obtain a 0.1 mol L -1 HCl solution. 200 µmol L -1

FcMeOH was prepared in 0.2 mol L -1 phosphate buffer (pH 6.7) with sonication for at least 2 hours to complete solubilization.

Microelectrode and microchip fabrication

The microfabrication process, developed in our lab, has been previously described [START_REF] Gouyon | Characterization of home-made graphite/PDMS microband electrodes for amperometric detection in an original reusable glass-NOA®-PDMS electrophoretic microdevice[END_REF]. Briefly, for the electrode fabrication, PDMS substrates containing microchannels of desired width are prepared by soft-lithography technic. A pattern is molded by UV exposure of spin-coated SU-8 on a silicon wafer with a photomask containing the design of the electrodes. Electrode pattern were printed on Kimolek® Paper with a Filmstar® photoplotter (Bernier Electronik, France).

PDMS is spread on the surface of the mold prior to degassing and reticulating at 70°C for 1h. A The dimensions of the channels and of the electrode as well as the estimation of the depletion yield were first optimized by performing numerical simulations using COMSOL Multiphysics 5.3, through a similar approach as previously described [START_REF] Oliveira | Development of a flow microsensor for selective detection of nitric oxide in the presence of hydrogen peroxide[END_REF]. Briefly, a laminar flow was considered in a rectangular microchannel by assuming a parabolic velocity profile in a twodimension. The electrochemical reactions were supposed to be limited by mass transport and the diffusion-convection equation was solved numerically by finite elements. As a boundary condition, concentration of the analyte was imposed to 0 mol L -1 at the polarized electrode surface. The diffusion coefficient of the analyte was assumed equal to 7.6.10 -6 cm 2 s -1 , which is the one of FcMeOH used as a model [START_REF] Cannes | Cyclic voltammetry and scanning electrochemical microscopy of ferrocenemethanol at monolayer and bilayer-modified gold electrodes[END_REF]. Simulations provided an optimal size of WE DEP versus the channel height, within the range of flow velocities that could be used experimentally.

According to the configuration and geometry of the microdevice described in Figure 1, depletion yields up to 95 % were expected for flow rate lower than 2 µL min -1 . The device was then applied to the recovery of Pd(II) and Au(III).

Results and discussion

Electrochemical behavior of FcMeOH under continuous flow

The electrochemical behavior of FcMeOH has been studied in the present configuration by CV on both WEs without flow (static mode) and under continuous flow (from 1 to 5 µL min -1 ), as

shown on Figure 2. The oxidation current for both WEs (WE DEP and WE det ) increase with the flow rate due to convection induced by continuous flow, leading to a steady-state mass transport to the electrodes and the observation of limiting currents. As expected in convective regimes, limiting currents increase linearly with the cubic root of flow rate (see inserts). At the highest flow rate (5 µL min -1 , i.e. 2.1 mm s -1 ), the faradic current started from about 0.1V and reached a limiting value only for potential values higher than 0.5V due mainly to ohmic drop. According to these voltammograms the potential on both electrodes should be comprised between +0.5 and +1.0 V to ensure a constant oxidation current under continuous flow (for 1 to 5 µL min -1 ) for optimal operating conditions. Indeed, to guarantee an optimum depletion efficiency (see sections below), the process needs to be performed at potential values that do not lead to a drastic current variation.

FcMeOH depletion

Cyclic voltammograms (CV) were performed at WE det with and without polarization of WE DEP at a constant potential of +1.0 V. Figure 3-a shows the mean values of limiting currents, noted as I lim,WEdet , monitored under these conditions at WE det as a function of flow rate. The depletion yield (Figure 3-b) was determined from these data by calculating the ratio between limiting currents at WE det (corrected from residual currents) with and without polarization of WE DEP :

eq.1

As expected theoretically, the depletion yield decreases by increasing the flow rate: the higher the flow, the shorter the residence time of redox species near the depletion electrode. However, experimental depletion yields were lower (by a factor ranging between 1.2 to 2) than the ones predicted by simulation, as shown in Figure 3-b. The depletion process was also evaluated by chronoamperometry (CA). More precisely, consecutive potential steps (ON) and (OFF) were performed at WE DEP , while maintaining WE det at a constant potential of 1 V for oxidizing remaining FcMeOH (Figure 4). Note that polarization of WE DEP , at 1 V and 0 V corresponds to the activation (ON) and deactivation (OFF) of the depletion process, respectively. As expected, polarization of WE DEP at +1,0V leads (i) to the increase of currents characteristic of FcMeOH oxidation and (ii) to a decrease of currents at WE det due to FcMeOH depletion. With this configuration of device and electrode material, the time delay to reach steady-state currents was estimated about 10 s. Then, similar experiments were conducted on a longer time scale. Figure 5-a shows chronoamperograms obtained at WE det for low flow rate (1 µL min -1 , i.e. 0.4 mm s -1 ) with or without polarization of WE DEP . First, a potential of 0 V was applied to WE det during 30 s for stabilization of the baseline followed by a potential step to 1.0 V during 60 s. This procedure was applied either in presence of FcMeOH or in PBS to consider the influence of residual currents into the evaluation of the charges developed at WE det , noted as Q WEdet . Experiments were also carried out for other flow rates. Thus In comparison to data obtained by CV (Figure 3-b) the same behavior was observed as a function of flow rate. A maximal depletion yield of 76.4% at the lowest flow rate 1 µL min -1 . The discrepancy between experimental and simulated data was similar. Indeed, the experimental depletion yields were lower than the predicted ones with: 76.4±3.3 % versus 99.6 at 1 µL min -1 and 41.1±7.0 % versus 73.4 % at 4 µL min -1 . As for data obtained by CV, this can be attributed to the composite material C-PDMS of the electrodes that does not lead to a well-defined active surface, perfectly planar, homogeneous, with no cavity or deformation. As shown by SEM [START_REF] Gouyon | Characterization of home-made graphite/PDMS microband electrodes for amperometric detection in an original reusable glass-NOA®-PDMS electrophoretic microdevice[END_REF],

the C-PDMS electrodes cannot be considered as planar for simulations. Also, the ohmic drop can reduce the performances of the depletion process if steady-state current at WE DEP , related to mass-transport limitation, is not reached at the applied potential. The larger the WE DEP ; the higher the influence of ohmic drop on depletion yield. However, according to our previous experiments, the potential of 1.0 V that was applied at both electrodes was sufficiently high to overcome the ohmic drop and to implement a process fully limited by mass transport. Therefore, only surface defaults and inactive areas of the C-PDMS electrodes could explain the deviations observed from numerical simulations.

Compared to the previously mentioned literature [START_REF] Oliveira | Development of a flow microsensor for selective detection of nitric oxide in the presence of hydrogen peroxide[END_REF], performance of this microdevice is lower.

In our case, the electrode material and aspect lead to significant differences since simulation did not consider the exact shape and nature of the material, and thus cannot reach the same level of efficiency than other metal electrode material. But since this system is low fabrication cost, C-PDMS electrode material is of interest to counteract the onerous and technically laborious development of platinum electrodes. Also the eventual electrode passivation after successive runs and its potential pollution are overcompensated by the originality of this reversible system.

Indeed, in case of major pollution of the electrodes during depletion processes, the device can be re-used as many times as portions of the band electrodes have not been exposed to the electrolyte solution. This is clearly an advantage compared to previously developed irreversibly closed depletion systems,In the case of FcMeOH study, no passivation was observed for the duration of the study on the different electrode areas used as continuous monitoring of blank samples was performed between each analysis so as to check the electrode and eventually renew the electroactive surface to avoid potential deterioration of the system.

Electrochemical reduction of chlorocomplexes of gold and palladium

The electrochemical activity of the different metal complexes has been previously examined on glassy carbon electrode in HCl solution [START_REF] Komsiyska | Electrocrystallization of Au nanoparticles on glassy carbon from HClO 4 solution containing [AuCl 4][END_REF][START_REF] Chang | Electrodeposition Fabrication of Pd Nanoparticles on Glassy Carbon Electrode Towards Methanol Oxidation Application[END_REF], with electrodeposition occurring for potential value of 0 V for Pd and 0.35 V for Au. Pd and Au in HCl solution can be reduced and metals can be deposited according to the following reactions:

(eq.3)

(eq.4)

Their electrodeposition on C-PDMS microelectrode within a microchannel occurs similarly but at different potentials (about -0.3 V and -0.5 V/C-PDMS for Au and Pd, respectively, Figure 6). The difference in values is due to the use of the pseudo-reference C-PDMS and to the nature of the C-PDMS electrodes. The stripping processes take place at around 0.26 and 0.30 V/C-PDMS for Au and Pd respectively, with behaviors similar to those described on glassy carbon electrodes. At 1 µL min -1 , the voltammograms on both WE DEP and WE det for Au and Pd do not show a waveshaped signal, except for Au on WE det (Figure 6-a). Indeed, the presence of a metal deposit on C-PDMS electrode such as Pd catalyzes hydroxonium ions reduction, leading to the overlapping of reduction signals and possibly to some interferences, as it will be discussed later.

Au and Pd electrodeposition as a function of applied potential and flow rate

Experiments were performed by chronoamperometry within the microchannel under constant flow rate (1 µL min -1 ) for different potentials. The deposition yields of both metals were evaluated separately. Three potential steps were successively applied at WE DEP and at WE det : (i) 0 V for 30 s to evaluate the current baseline, (ii) a constant potential between -0.4 and -1.2 V for 60 s to reduce metal ions and finally (iii) +0.5 V for more than 100 s to fully oxidize the metal deposits.

Figure 7 shows the chronoamperograms obtained at WE det with the corresponding oxidation charges (third step) at both WE DEP and WE det as a function of the applied potential for metal reduction. The depletion yield as a function of flow rate for both metals is given in Figure 8. The same experiment than figure 7 was performed but without activation of WE DEP so as to estimate the depletion yield, calculated from the oxidation charges Q developed at WE DEP and WE det during the third step as follows:

eq.5

Note that the calculation of the depletion yield was not based on cathodic charges because the metal reductions are prone to interferences from solvent reduction. The highest depletion yields were obtained for the lowest flow rates, as previously observed for FcMeOH, namely 68.2 and 44.6 % for Au and Pd, respectively at 1 µL min -1 . WE DEP is set at the same potential than WE det when activated.

Depletion yield as a function of metal concentration and mechanism of deposition

The previously described results allowed setting the optimum parameters for the depletion of precious metals as follows: reduction potential values of -1.0 and -1.2 V for Au and Pd, respectively and minimal flow rate of 1 µL min -1 . The evaluation of the depletion yield as a function of the metal concentration in acidic solution was then performed (Figure 9) using the same procedure as in Figure 8 for concentrations ranging between 25 and 500 µmol L -1 for Au and for concentrations ranging between 25 to 1000 µmol L -1 for Pd. The data showed that the depletion yield decreased with the increase in concentration for both metals, with values of up to 89% for Pd (25 µmol L -1 / 2.7 mg L -1 ) and 71 % for Au (25 µmol L -1 / 4.9 mg L -1 ). These results are promising since they are obtained for concentrations close to the expected ones in real leach liquors from waste printed circuits boards recycling processes (9.4 to 94.0 mg L -1 for Pd and 5.1 to 50.8 mg L -1 for Au) [START_REF] Kim | Selective recovery of gold from waste mobile phone PCBs by hydrometallurgical process[END_REF].

Simulations performed under similar conditions predicted a depletion yield of 89% at 2.6 µL min - 1 , which is higher than the experimental one. The difference between theoretical and experimental values can be attributed to several factors as discussed for FcMeOH, but other issues can also affect the process in the case of the metal deposition. Indeed, for Au the deposition of the metal occurs through its reduction into Au(+I) (as described by eq. 4) followed by the formation of Au(0) by dismutation. This phenomenon occurs after adsorption of gold complexes onto active sites, leading to the formation of multiple entities assimilated to nanoparticles [START_REF] Komsiyska | Electrocrystallization of Au nanoparticles on glassy carbon from HClO 4 solution containing [AuCl 4][END_REF]. The deposition of Au occurs in parallel to the formation of Au(+III), thus a fraction of the sample doesn't turn into solid and it is removed from the system without any treatment and being recovered. For Pd, the mechanism of growth of metallic deposit is discussed in the literature since it does not seem to correspond to a simple model where the ion is reduced on existing nuclei sites [START_REF] Gu | Mechanism for nucleation and growth of electrochemical deposition of palladium(II) on a platinum electrode in hydrochloric acid solution[END_REF]. Depending on the applied potential, the growth of Pd can be described by different models [START_REF] Alvarez | Formation of Cu/Pd bimetallic crystals by electrochemical deposition[END_REF] and only for high overpotential reduction value, bulk Pd deposit is observed [START_REF] Sahin | Electrochemical Palladium Deposition for Reducing Critical Dimensions in Nanostructures[END_REF]. In our case, the applied potential during the reduction step of chronoamperometric experiment corresponds to high overpotential, which is in favor of the formation of bulk metals. Moreover, since this potential value is high, reduction of the solvent overlaps the metal reduction and this leads to loss of performances of the electrode, explaining the impossibility to reach high depletion yield. The fact is that as soon as the deposition process begins for a given metal, the formation of the metal on the C/PDMS electrodes can induce a change in the reduction of metal complexes. This was evaluated by measuring the mean current of reduction of each metal on C-PDMS electrode previously modified with the same metal deposit. As illustrated in Figure 10 In the case of Au, the current related to the reduction of the metal complex is higher on Aumodified electrode than on C-PDMS for all the considered potential range. This can be attributed to a higher active surface or to a thermodynamically more favourable deposition on Au substrates. Thus, thin layered gold modification of C-PDMS electrode material is recommended to enhance the performances of the process. In the case of Pd, the current related to Pd reduction is clearly lower on Pd-modified electrode than on C-PDMS electrode for all the considered potential range. This can be explained by the fact that Pd acts as a catalyst at such a high reduction potential for the hydroxonium reduction. The reduction of Pd is thus overlapping with that of protons. This leads to a decrease of the deposition rate and potentially explains why the depletion yield cannot be larger. All these results demonstrate also why the depletion yield for Au is much higher than for Pd (Figure 8).

Conclusion

The depletion of precious metals on home-made reversible microchips was demonstrated with the integration of graphite/PDMS microelectrodes within microfluidic channels. After characterization of process performances with a model electroactive probe (FcMeOH), this device was applied for the electrodeposition of Au and Pd, with a depletion yield of up to 89%

for Pd and 71% for Au. These values were obtained for low metal concentration (25 µmol L -1 ), which is in the range of those expected from leach liquors of PCBs wastes, undergoing a hydrometallurgical treatment. In order to further increase the depletion yield, other experimental parameters can be explored, such as the electrode size so as to increase the overall active surface and to overcome surface defaults. Such a microchip thus opens the way for real leach liquor treatment from the waste electric equipment recycling industry or for wastewater purification. To do so, a focus is therefore recommended on the optimization of the confinement on which this process is based, so as to increase the depletion yield. Indeed, this setup can be implemented on industrial scale provided that the confinement is ensured and optimized. It can also be used as a promising tool for the depletion of contaminant species whose removal rests on their electrochemical reactivity, such as organic pollutants or inorganic ions.

  mix of graphite powder and PDMS (C-PDMS, 1:1, w/w) is spread in the microchannels and polished by hand to remove the excess. The resulting electrodes are placed in an oven at 70°C for 3h before use. For the microchannel fabrication, a PDMS mold containing the positive pattern is prepared by the same technic. The mold is applied on top of a glass substrate previously covered with NOA ® 81. After UV exposure, the open-channel is revealed by properly demolding the PDMS mold. The two part are then assembled so as to close the chip, forming a spontaneous conformal contact between the open-channel (NOA81 ® ) and the electrode (C-PDMS on PDMS substrate). The scheme and a picture of the setup are shown in Figure1. In such a device, WE DEP is the working electrode dedicated to the depletion process (and also to the recovery of the metals), WE det is the working electrode dedicated to the determination of the considered species and thus to the evaluation of the recovery efficiency.

Figure 1 -) Photography of the experimental setup 2 . 4 .

 124 Figure 1 -(a) Scheme of microchannel with four integrated C/PDMS microbands electrodes. WE DEP is dedicated to the depletion process (and thus to the recovery), WE det is dedicated to the determination of the considered species and thus to the evaluation of the recovery efficiency. Dimensions in μm (unless specified). * upper part of the setup (NOA81 ® microchannel; ** lower part of the setup (C-PDMS electrodes in PDMS substrate) (b) Photography of the experimental setup 2.4. Electrochemical measurement and depletion

Figure 2 -

 2 Figure 2 -Cyclic voltammograms of 200 µmol L -1 FcMeOH in 0.2 mol L -1 phosphate buffer at (a) WE DEP and (b) WE det , in static mode (full line) and under continuous flow (from 1 to 5 µL min -1 ). Scan rate: 25 mV s -1 . Inserts correspond to the plots of limiting currents as a function of cubic root of flow rate.

Figure 3 -

 3 Figure 3 -(a) Limiting currents measured by cyclic voltammetry (from data in Fig. 2) at WE det without (OFF) and with (ON) polarization of WE DEP . (b) Corresponding depletion yield evaluated from eq.1 as a function of flow rate. WE DEP is biased at 1.0 V when polarized (ON).

Figure 4 -

 4 Figure 4 -(a) Potential steps applied to WE DEP for successive activations (1 V) and deactivations (0 V) of the depletion process. WE det is continuously polarized at 1 V. (b) Corresponding chronoamperograms monitored at WE det (full line) and WE DEP (dashed line). 200 µmol L -1 FcMeOH in 0.2 mol L -1 phosphate buffer and flow rate of 1 µL min -1 .

Figure 5 - 2 Figure 5 -

 525 Figure 5 -(a) Chronoamperometry at WE det (30 s at 0 V and 60 s at 1.0 V) without depletion (curves 1 and 2, WE DEP OFF) and with depletion (curves 3 and 4, WE DEP polarized at 1 V) at 1 µL min -1 flow rate. (b) Experimental charge Q WEdet developed at WE det during the second step as a function of flow rate. (c) Depletion yield evaluated by eq. 2 from experimental data (cross points, dashed line) and simulations (solid curve) as a function of flow rate (200 µmol L -1 FcMeOH in 0.2 mol L -1 phosphate buffer).

Figure 6 -

 6 Figure 6 -Cyclic voltammograms of (a) 500 mol L -1 Au in 0.14 mol L -1 HCL and (b) 1000 mol L -1 Pd in 0.1 mol L -1 HCl on WE det and WE DEP at 25 mV s -1 (static mode)

Figure 7 -

 7 Figure 7 -(a) Chronoamperograms (i & ii) of 1000 mol L -1 Pd in 0.1 mol L -1 HCl and 500 mol L -1 Au in 0.14 mol L -1 HCL at WE det under 1 µL min -1 flow rate with WE DEP polarized at various potential and (b) oxidation charges corresponding to the third step measured at WE det and on WE DEP as a function of the reduction potential applied to WE DEP during the second step.

Figure 8 -

 8 Figure 8 -Depletion yield estimated from Eq. 5 for Pd and Au as a function of the flow rate. ( 500 µmol L -1 Au in 0.14 mol L -1 HCl, 1 mmol L -1 Pd in 0.1 mol L -1 HCl, 200 µmol L -1 FcMeOH in phosphate buffer 0.2 mol L -1 , first step (0 to 30 s): WE det = 0 V/C-PDMS; second step (30 to 90 s): WE det = -1 V, -1.2 V and +1 V/C-PDMS for Au, Pd and FcMeOH, respectively, and third step (90 to 200 s): WE det = 0.5 V/C-PDMS (for Pd and Au, only). WE DEP is set at the same potential than WE det when activated.

Figure 9 -

 9 Figure 9 -Depletion yield of Au and Pd as function of their concentration for flow rate of 1 µL min -1 . Same conditions as in Figure 8.

  , the mean current of metal ion reduction obtained by chronoamperometry on C-PDMS or on metal modified C-PDMS electrode are represented as a function of the applied potential (with blank subtraction).

Figure 10 -

 10 Figure 10 -Mean current value obtained by chronoamperometry as a function of the potential for (a) 1000 mol L -1 Pd in 0.1 mol L -1 HCl and (b) 500 µmol L -1 Au in 0.14 mol L -1 HCl at WE DEP C-PDMS electrode (open square) and WE DEP C-PDMS modified electrode (solid square). Metal deposits were performed at -1 V/C-PDMS on WE DEP for 120s without flow. The same experiments with WE det let to similar results (data not shown).

Acknowledgements

This work was supported by the French Environment and Energy Management Agency (ADEME) and the Chaire "Mines Urbaines" from ParisTech foundation, supported by Ecosystèmes. This work has received support of "Institut Pierre-Gilles de Gennes" (Laboratoire d'excellence: ANR-10-LABX-31, "Investissements d'avenir": ANR-10-IDEX-0001-02 PSL and Equipement d'excellence: ANR-10-EQPX-34).


Arnaud Huguet

Rime Sylvain Bernard

El Khatib

Martina I Gocke

Guido L B Wiesenberg

Sylvie Derenne

Multiple stages of plant root calcification deciphered by chemical and micromorphological analyses

Keywords: rhizoliths, SEM, 13 C NMR, loess sediments, secondary carbonates, organic 23 matter, paleoenvironment 24 25 26

come   L'archive ouverte pluridisciplinaire

3 53 as root-derived biopores down to several meters depth below the recent soil or associated 54 paleosol. The post-sedimentary incorporation of root-derived OM can lead to a large overprint 55 of the initial signal in loess sediments, as shown for example for lipid biomarkers (e.g. n-56 alkanes, fatty acids, GDGTs; [START_REF] Huguet | Root-associated 572 branched tetraether source microorganisms may reduce estimated paleotemperatures in 573 subsoil[END_REF][START_REF] Gocke | Lateral and depth variation of loess organic 555 matter overprint related to rhizoliths revealed by lipid molecular proxies and X-ray 556 tomography[END_REF]. The presence of 57 carbonate rhizoliths in loess settings can therefore bias paleoenvironmental reconstructions.

58 Corresponding data have to be interpreted with caution, as only limited knowledge about their 59 formation mechanisms is available (e.g. Klappa, 1980;Gocke et al., 2011Gocke et al., , 2014a, b;, b;Li et al., 60 2015b;Sun et al., 2019a;[START_REF] Brazier | 514 Multi-isotope approach (δ44/40Ca, δ88Sr and 87Sr/86Sr) provides insights into rhizolith 515 formation mechanisms in terrestrial sediments of Nussloch (Germany)[END_REF].

61

It is generally assumed that root fossilization is controlled or induced by complex organic-62 inorganic interactions at the plant level (Klappa, 1980;[START_REF] Jaillard | Structure and composition of calcified roots, and 578 their identification in calcareous soils[END_REF]Kautz et al., 2013; 63 [START_REF] Zamanian | Pedogenic carbonates: Forms and 674 formation processes[END_REF][START_REF] Brazier | 514 Multi-isotope approach (δ44/40Ca, δ88Sr and 87Sr/86Sr) provides insights into rhizolith 515 formation mechanisms in terrestrial sediments of Nussloch (Germany)[END_REF], but the exact processes leading to the formation 64 of rhizoliths remain poorly constrained. Here, we report the chemical and micromorphological 65 characterization of roots at different stages of fossilization that have been sampled at different 66 depths (2.15 m, 3.5 m, 4 m and 8 m depth) from the extensively studied Late Pleistocene 67 loess-paleosol sequence of Nussloch (SW Germany) (e.g. [START_REF] Antoine | Rapid and cyclic aeolian deposition during the Last Glacial in European loess: a 496 high-resolution record from Nussloch, Germany[END_REF]Gocke et al., 68 2011;[START_REF] Zech | Reconstruction of the late 679 Quaternary paleoenvironments of the Nussloch loess paleosol sequence, Germany, using n-680 alkane biomarkers[END_REF][START_REF] Huguet | Root-associated 572 branched tetraether source microorganisms may reduce estimated paleotemperatures in 573 subsoil[END_REF]. The samples were chosen on the basis of their 69 representativeness of the rhizoliths encountered along the Nussloch profile. ,Nussloch,SW Germany (49.19°N,8.43°E,83 217 m above sea level), as described in detail by [START_REF] Gocke | Rhizoliths in loess -541 evidence for post-sedimentary incorporation of root-derived organic matter in terrestrial 542 sediments as assessed from molecular proxies[END_REF]Gocke et al. ( , 2011Gocke et al. ( , 2014a, b), b). The 84 sampled profile comprises 13.1 m of loess and soil units, including the recent soil, with 3 85 main paleosols and 14 incipient paleosols [START_REF] Gocke | Lateral and depth variation of loess organic 555 matter overprint related to rhizoliths revealed by lipid molecular proxies and X-ray 556 tomography[END_REF]. At the top of the profile, the 86 recent soil is developed as 0.6 m thick Vertic Cambisol Calcaric Siltic [START_REF] Gocke | Differentiation of plant derived 548 organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF].

87 The present vegetation, related to living roots, consists of natural broad-leaf forest and non-88 native robinia (Robinia pseudoacacia) as well as smaller shrubs and herbaceous plants and 89 was described in detail by [START_REF] Gocke | Differentiation of plant derived 548 organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF]. It should be noted that present plant species 90 were most likely not the dominant plants over the past decades or centuries due to former 91 agricultural use. Rhizoliths at Nussloch were formed during various phases throughout the 92 Holocene, with ages between 3 and 10 kyr [START_REF] Pustovoytov | An isotopic study of a late Quaternary loess-paleosol 640 sequence in SW Germany[END_REF]Frechen et al., 93 2007;Gocke et al., 2011) and were derived from roots of unknown trees or shrubs (likely 94 hazel, oak, beech and alder; [START_REF] Gocke | Differentiation of plant derived 548 organic matter in soil, loess and rhizoliths based on n-alkane molecular proxies[END_REF] 141 NMR without any pre-treatment. In contrast, rhizoliths were treated with 3M HCl to remove 142 carbonates which would interfere with the organic carbon signal during analysis, rinsed with 143 ultrapure water and then frozen at -20 °C before being freeze-dried [START_REF] Huguet | Root-associated 572 branched tetraether source microorganisms may reduce estimated paleotemperatures in 573 subsoil[END_REF].

144 Samples were crushed in a ball mill before analysis.

145 Solid state 13 C cross polarization magic angle spinning nuclear magnetic resonance (CP 146 MA NMR) was performed on a Bruker Avance 500 at 125 MHz for 13 C using 4 mm diameter 147 zircon rotors. Cross polarization allows the magnetization transfer from 1 H to 13 C, leading to 148 an increase in the signal/noise ratio. Magic angle spinning at 14 kHz was used to reduce 149 chemical shift anisotropy and to average dipolar interactions, hence decreasing the linewidths.

150 The contact time between 13 C and 1 H in the CP MAS sequence was set to 1 ms and the 151 repetition time to 1 s. The NMR spectra were decomposed and integrated using Dmfit 152 software -version 20150521 [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR 619 spectra[END_REF]. The subpeaks resulting from the 153 decomposition were further grouped within 6 main regions based on existing literature (e.g. 165 were observed within the Nussloch loess-paleosol sequence, as shown for example at a depth 166 of 3.5 m (Fig. 1a). It was previously reported that rhizoliths with diameters ≥ 10 mm were 167 predominant between 2 m and 6 m depth and rhizoliths with diameters < 10 mm were mainly 168 observed below 6 m [START_REF] Gocke | The present SEM results further show for the first time that, 184[END_REF]. Within some of the rhizoliths, the central part of 169 the rhizolith is hollow (Fig. 1b), indicating the absence of carbonates filling the void. Even 170 though some authors tried to classify root-related structures (Klappa, 1980) or more generally 171 secondary carbonate accumulations (Barta, 2011) in terrestrial settings based on their external 172 shapes, it remains difficult to classify the diversity of calcified root structures encountered in 173 Nussloch. The general term "rhizolith" will be used here in a broad sense to refer to root 174 traces, i.e., organo-mineral structures formed through fossilization of former roots.

175 Typical rhizoliths from Nussloch with diameters of 10-20 mm were analyzed for their 176 internal structure. Independent of the depth, Scanning Electron Microscopy (SEM) revealed 177 the presence of numerous small, fine roots encrusted by secondary carbonates within each 178 rhizolith (e.g. Fig. 1c, f; Supp. Fig. 1). and microscopic analyses of initially calcified roots (Fig. 2d,e,f). The initiation of the 363 calcification is directly related to rhizosphere processes and to the associated high 364 microbial activity [START_REF] Jones | Organic acids in the rhizosphere -a critical review[END_REF]. Roots increase acidity in the surrounding 365 soil/sediment by releasing CO 2 and exudates, which contains especially organic acids 366 (Klappa, 1980). Primary CaCO 3 from soil/sediment is dissolved and Ca 2+ is being 367 transferred in solution towards the root. At the root surface not all calcium, magnesium, 368 strontium and other elements might be taken up by the plants as nutrients and can be 369 precipitated as secondary carbonates at the root surface (Barta, 2011). This is supported 370 by the high CO 2 concentration in the vicinity of the root due to root and microbial 371 respiration [START_REF] Hinsinger | Origins of root-mediated pH changes 566 in the rhizosphere and their responses to environmental constraints: a review[END_REF]. In addition, Ca 2+ and Sr 2+ from topsoils can be 372 taken up by plant roots and translocated towards larger depths in loess-paleosol 373 sequences as described by [START_REF] Brazier | 514 Multi-isotope approach (δ44/40Ca, δ88Sr and 87Sr/86Sr) provides insights into rhizolith 515 formation mechanisms in terrestrial sediments of Nussloch (Germany)[END_REF]. Simultaneously, or after precipitation 374 of the carbonate coating on the surface of the fine roots, surplus calcium can be 375 precipitated as calcium carbonate within the cortex cells when fine roots are still alive, 376 as observed in Nussloch (Fig. 2g). Some of the fine roots may be totally infilled by 377 calcium carbonate, with precipitation around phloem and even xylem cells, as seen for [START_REF] Strand | Irreconcilable 647 differences: fine root lifespans and soil carbon persistence[END_REF][START_REF] Ding | Fine root longevity and below-527 and aboveground litter production in a boreal Betula pendula forest[END_REF] and an apparent turnover of eight years 393 [START_REF] Fröberg | Residence time of fine-root carbon using radiocarbon measurements of 535 samples collected from a soil archive[END_REF], microrhizoliths are likely formed over a short period of time (~ 1 394 year).

F o r R e v i
395

3) Concomitantly or subsequently to the previous steps, calcium carbonate may also 396 precipitate in voids between several of the fine roots. This is supported by the presence 397 of a large number of microrhizoliths within one "large" rhizolith from Nussloch with 398 precipitated carbonate in-between individual microrhizoliths (Fig. 1c, f; Supp. Fig. 1).

399

The agglomeration of microrhizoliths may be seen as a hotspot for nutrient and water 400 acquisition as well as microbial activity [START_REF] Jones | Organic acids in the rhizosphere -a critical review[END_REF] [START_REF] Huguet | Root-associated 572 branched tetraether source microorganisms may reduce estimated paleotemperatures in 573 subsoil[END_REF]. Especially in deeper parts of the soil, biopores 405 often serve as places for renewed root growth [START_REF] White | The distribution and abundance of wheat roots in a 666 dense, structured subsoil-implications for water uptake[END_REF]. After the 406 death of the plants, large biopores formed by decaying roots may remain stable for 407 millennia in fine-textured sediments such as loess (Kerényi, 2015). Also in the profile 408 of Nussloch many biopores were observed throughout the whole sequence, i.e. in depth 409 intervals with high and low abundance of recent roots and rhizoliths, respectively 410 [START_REF] Gocke | The present SEM results further show for the first time that, 184[END_REF]. Consequently, it is likely in the Nussloch sequence that 411 agglomeration of microrhizoliths occurs in biopores that were primarily produced by 412 large tap-roots of shrubs and trees. Either during the decay of these large roots or at one 413 point thereafter while still biopores were preserved, fine roots colonized the large 414 biopore or decaying root, and subsequently formation of microrhizoliths occurred by 415 the precipitation of calcium carbonate with only traces of Mg or Si (Fig. 3), whereas 416 minerals like quartz, micas or feldspars derived from primary loess were only detected 417 close to the outer rims of large rhizoliths (Gocke et al., 2011). Of course, it is possible 418 that several generations of microrhizoliths can develop in one biopore of a diameter of 419 several cm, while some older microrhizoliths might serve as additional source of 420 nutrients on top of the surrounding sediment [START_REF] Brazier | 514 Multi-isotope approach (δ44/40Ca, δ88Sr and 87Sr/86Sr) provides insights into rhizolith 515 formation mechanisms in terrestrial sediments of Nussloch (Germany)[END_REF]. Thus, the carbonate 421 remains might be partially destructed as indicated by the different status of 422 microrhizoliths within one large rhizolith, with (i) almost intact microrhizoliths 423 presenting perfectly round shaped structures, (ii) partially calcified microrhizoliths and 424 (iii) residual microrhizolith structures with no clear internal structure anymore (Fig. 1).

425

The assemblage of numerous microrhizoliths in biopores left behind after decay of former 426 larger roots led to the formation of larger rhizoliths, might have occurred during a large time 427 span starting with the development of large roots and biopores after their decay shortly after Chemical shift (ppm)

3 .

 3 [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial 598 residues as inputs to soil organic matter[END_REF][START_REF] Lemma | Decomposition and 607 substrate quality of leaf litters and fine roots from three exotic plantations and a native 608 forest in the southwestern highlands of Ethiopia[END_REF][START_REF] Mathers | 13 C-NMR analysis of 626 decomposing litter and fine roots in the semi-arid Mulga Lands of southern Queensland[END_REF]: alkyl C = 0-45 ppm 155 (includes aliphatic and α-amino C); O/N-alkyl C = 45-90 ppm (includes methoxyl C and C in 156 amino groups); 90-110 pm = di-O-alkyl C (includes anomeric C in carbohydrates); 110-140 157 ppm = aromatic and unsaturated C; 140-160 ppm = phenolic C; 160-220 ppm = carbonyl C158 (includes carboxylic C, amides, ketones/aldehydes). More details regarding 13 C NMR and its 159 application to soil organic matter characterization can be found elsewhere (e.g.Knicker and 160 Nanny, 1997;[START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial 598 residues as inputs to soil organic matter[END_REF][START_REF] Chukov | Application of 13C NMR spectroscopy to 520 the study of soil organic matter: A review of publications[END_REF]. 161 162 Results and discussion 163 3.1. Microscopic characterization of rhizoliths 164 Rhizoliths of largely variable sizes (a few cm to m long) and diameters (a few mm to cm)

  Scanning electron micrographs showing the structure of rhizolith collected at 2.15 m depth in cross section. The zone highlighted in red in panel (a) was enlarged for better visualization of the rhizolith internal structure (cf. panel (b. 2. 13 C NMR spectrum of rhizolith collected at 2.15 m depth in Nussloch (SW Germany).

  Root and rhizolith samples were impregnated with epoxy resin, cross-sectioned 131 perpendicular to their longitudinal direction, polished and gold-coated for scanning electron 132 microscopy (SEM) investigations using a Zeiss Sigma microscope operating at the 133 Laboratoire de Geologie (ENS-Paris). Backscattered electron imaging (BEI) was carried out 134 at 10kV and a working distance of 9.4 mm using an AsB detector. Energy Dispersive X-ray 135 Spectrometry (EDXS) micro-analyses were performed using INCA SmartMapTM software.136 Elements typically encountered in secondary carbonates and surrounding loess (Ca,O, Al, 137 Mg, Si) were traced in this study.

	129	2.2	Electron microscopy
	130
			F F
	138		o r o r
	139	2.3	Solid state 13 C NMR	R R
				e v i e w e v i e w
				. They occur continuously from the recent soil O O
	95 to 8.8 m depth, with frequencies reaching up to 196 m -2 at 2.55 m, and are also present below n l n l
	96 10 m underneath a major paloeosol (Lohne Soil) in lower frequencies (≤ 12 m -2 ; Gocke et al., y y
	97 2014a). They can be found in various shapes and sizes (diameter between a few mm to ca. 5
	98 cm and length from mm to several m; Gocke et al., 2011).
	99	Three types of samples were collected in Nussloch for comparison of the organo-mineral
	100 structure of the roots: (i) present vegetation (including Robinia pseudoacacia); (ii) initially
	101 calcified roots and (iii) rhizoliths. Initially calcified roots correspond to living roots with a
	102 thin (< 1 mm) powdery carbonate coating on their surface. In contrast, samples termed as
	103 "rhizoliths" are fully carbonated roots with scarce or no root remains visible to the naked eye.
				6

140

Roots from present vegetation and those with a thin carbonate crust were analyzed by 13 C

  was observed for initially calcified roots to occur first around the fine 454 root, the epidermis acting as a first barrier, and then within the root, within the cortex cells 455 and even sometimes around the phloem and within the xylem. Fossilization of large and fine

	453 secondary carbonates 456 roots by secondary carbonates was shown to allow a good preservation of root cell structure
	457 and selective enrichment of recalcitrant root biopolymers such as lignin and suberin,
	458 suggesting that the formation of (micro)rhizoliths may be initiated during the lifetime of the
	459 root and continues after its death. This has to be systematically determined before using
	460 (micro)rhizoliths for paleoenvironmental or paleoecological reconstructions and interpreting
	F F 461 the corresponding data with confidence. Lipid (e.g. n-alkanes, GDGTs, fatty acids) and F F F F F
	o r o r 462 compound-specific isotope analyses (including radiocarbon dating) could be used to better o r o r o r o r o r
	463 constrain this context.	R R R R R R R
	464 465 466 467	e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w
	468 469 470 471 472 473 474	O n l y O n l y O O O O O n l n l n l n l n l y y y y y
	475
	476
	477
	478
	479
	480
	481
	482
	483
	484
	485
	486
	487
	488
	489
		19