Satish Bodakuntla

Carsten Janke
email: carsten.janke@curie.fr

Maria M Magiera
email: maria.magiera@curie.fr

Knocking out multiple genes in cultured primary neurons to study tubulin posttranslational modifications

Keywords: Primary cells, primary hippocampal neurons, microtubules, tubulin posttranslational modifications, polyglutamylation, mouse models

Microtubules, as integral part of the eukaryotic cytoskeleton, exert numerous essential functions in cells. A mechanism to control these diverse functions are the posttranslational modifications of tubulin. Despite known for decades, relatively little insight into the cellular functions of these modifications has been gained so far. The discovery of tubulin-modifying enzymes and a growing number of available knockout mice now allows working with primary cells from those mouse models to address biological functions and molecular mechanisms behind those modifications. However, a number of those mouse models show either lethality or sterility, making it difficult to impossible to obtain a sufficient number of animals for a systematic study with primary cells. Moreover, many of those modifications are controlled by several redundant enzymes, and it is often necessary to knock out several enzymes in parallel to obtain a significant change in a given tubulin modification. Here we describe a method to generate primary cells with combinatorial knockout genotypes using conditional knockout mice. The conditional alleles are converted into knockout in the cultured primary cells by transduction with a lentivirus encoding cre-recombinase. This approach has allowed us to knock out the two main brain deglutamylases in mouse primary neurons, which leads to strongly increased polyglutamylation in these cells. Our method can be applied to measure different cellular processes, such as axonal transport, for which it can be combined with the expression of different fluorescent reporters to label intracellular proteins. Using a panel of conditional knockout mice, our method can further be applied to study the functions of a variety of tubulin modifications that require simultaneous knockout of multiple genes.

Introduction

Microtubules, together with actin and intermediate filaments constitute the cytoskeleton of each eukaryotic cell. Microtubules assemble from evolutionarily conserved aand b-tubulin heterodimers in a highly dynamic manner, and are involved in a plethora of cellular processes: they build meiotic and mitotic spindles during cell division, drive directed cell migration, determine cell shape, and serve as tracks for intracellular transport. Microtubules are particularly crucial in neurons, where they play a pivotal role in determination of neuronal polarity, dendritic arborization, axon branching and in the transport of cellular cargoes from the cell body to synaptic terminals and vice versa [1,2]. A key question is how microtubules are able to adapt their properties to participate in those highly diverse events. A mechanism to control microtubule properties that could potentially regulate these different functions are tubulin posttranslational modifications (PTMs), such as acetylation, detyrosination or polyglutamylation, which are all highly enriched in neurons [3].

Though the presence of multiple PTMs on brain tubulin was identified decades ago, their biochemical and functional characterisation became possible only after the enzymes catalysing them had been discovered (reviewed in [4]). Overexpression or knockdown of those enzymes allowed performing experiments at the cellular level thus providing first insights into the functions of tubulin PTMs (reviewed in [5,6]). In parallel, the development of a variety of mouse models lacking modifying enzymes delivered novel insights into the physiological roles of tubulin PTMs [7][8][9][10][11][12][START_REF] Grau | Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration[END_REF][START_REF] Gadadhar | Tubulin glycylation controls primary cilia length[END_REF][START_REF] Giordano | Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility[END_REF]. Approaches allowing to combine these mouse models with cell-based assays can bolster the discovery of underlying mechanisms in physiologically relevant cell types. For instance, culturing primary neurons from mouse models with altered modification levels has shed light on the role of tubulin PTMs in several neuronal functions [11,[START_REF] Silva | Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis[END_REF]7,8,[START_REF] Gilmore-Hall | CCP1 promotes mitochondrial fusion and motility to prevent Purkinje cell neuron loss in pcd mice[END_REF][START_REF] Marcos | Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone[END_REF]. Culturing primary cells with altered modification levels can be straightforward for some mouse models, and very difficult for others. Mice with no sterility phenotypes can be obtained from homozygous breeding pairs, which is the case for the two enzymes involved in tubulin acetylation, αTAT1/Mec-17 [START_REF] Akella | MEC-17 is an alpha-tubulin acetyltransferase[END_REF][START_REF] Shida | The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation[END_REF] and HDAC6 [START_REF] Hubbert | HDAC6 is a microtubule-associated deacetylase[END_REF]. Sterile mouse strains, or mice with perinatal lethality, such as the knockout for tubulin tyrosine ligase (TTL) [11] need to be bred from heterozygous progenitors, which subsequently requires single-embryo cell cultures followed by genotyping. More difficult still are the combinatorial knockout strains, in particular with one or more of the transgenes leading to sterility. These are almost impossible to use for generating sufficient amounts of cells, as the breeding schemes are very complex, and the relevant offspring is statistically too rare to perform systematic cell culture studies.

Here we propose a method to overcome this problem, which we have developed to study the role of tubulin polyglutamylation in neurons.

Polyglutamylation is generated by multiple enzymes belonging to the family of tubulin tyrosine ligase like proteins (TTLL) [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF] and removed by enzymes from the cytosolic carboxy peptidase (CCP) family [10,[START_REF] Tort | The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids[END_REF]. Several of these enzymes are involved in controlling polyglutamylation levels in neurons: TTLL1 and TTLL7 are the major glutamylases [8,[START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF], and CCP1 and CCP6 are the main deglutamylases [10,7]. To strongly reduce polyglutamylation in the brain, both TTLL1 and TTLL7 need to be depleted concomitantly, while to increase polyglutamylation, the inactivation of CCP1 and CCP6 is required in at least some parts of the brain, such as hippocampus [7]. In both cases, one of the two knockout mouse strains (CCP1 and TTLL1) is male sterile, and mice can thus only be bred heterozygous, which strongly reduces the probability of generating double-knockout mice.

To establish a reliable and sensitive cell biological approach for studying the impact of polyglutamylation on neuronal functions, we developed a cell-culture based method to achieve increased polyglutamylation in primary hippocampal neurons, which can be easily extended to study the effects of increased or decreased PTMs other than polyglutamylation.

There are two possible strategies allowing to obtain primary neurons with perturbed tubulin PTM levels: overexpressing or silencing the corresponding enzyme(s). To obtain increased polyglutamylation levels one can overexpress glutamylating enzymes, or deplete the main deglutamylating enzymes, either by RNA interference (RNAi) or by gene knockout.

Overexpression of glutamylases has many drawbacks, the most important being that the amount of enzyme is far from being physiologically relevant, and could thus result in artefacts. For instance, all glutamylases overexpressed in Hela cells promiscuously glutamylated aand b-tubulin, while normally these enzymes are specific to either aor b-tubulin [START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF]. Moreover, in neurons, the most relevant enzyme is the glutamylase TTLL1, which could so far not be expressed as an active enzyme in cells due to the necessity to form a complex with at least four other proteins [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF]. Finally, some tubulin-modifying enzymes have been observed to bind microtubules when overexpressed (our unpublished observations), which might alter microtubule dynamics and could impair the movement of organelles on the microtubules, independently of the PTM generated by the enzyme.

We thus strongly favour the strategy to remove the major deglutamylating enzymes, which in neurons are CCP1 and CCP6 [10,7]. We decided against RNAi, as it rarely generates a complete loss of the target transcript, which in the case of the low-expressing tubulinmodifying enzymes would be very hard to control. Thus, our method of choice was to culture primary neurons from knockout mouse strains. The breeding scheme we developed to obtain ccp1 -/-ccp6 -/-animals was very difficult for a number of reasons, such as the male infertility of ccp1 -/-mice and a general lack of fitness of the double-knockout mice (which is why we used Ccp6 flox/flox nestin-cre to breed the double-knockout animals [7]). The theoretical turnout of 1/16 of this breeding scheme was not appropriate for systematic studies using primary neuronal cultures, therefore we generated a mouse model in which both, CCP1 and CCP6, can be conditionally knocked out (Ccp1 flox/flox Ccp6 flox/flox ). These mice behave like wild type and thus can be easily bred to give homozygous embryos, which are the source of primary hippocampal neurons. Once neurons are cultured, they are transduced with a lentivirus expressing cre recombinase to convert both flox alleles into knockout alleles, leading to the removal of deglutamylases, which results in tubulin hyperglutamylation. In parallel, the neurons from the same culture are transduced with a control virus, and can be used as an internal control with normal polyglutamylation levels. To verify the effect of the cre recombinase alone, the equivalent experiment is performed using neurons from wild type embryos transduced with the same GFP-and GFP-cre viruses.

Thus, we describe a novel, physiologically relevant cell-based assay to attain excessive polyglutamylation and its possible applications. Our cell-culture based method is not limited to studying microtubules or neurons, but has broader applications in the field of cell biology.

As far as microtubule PTMs are concerned, our method can be expanded to study the impact of reduced polyglutamylation (using Ttll1 flox/flox Ttll7 flox/flox neurons) or reduced detyrosination (with Vash1 flox/flox Vash2 flox/flox neurons), all of which require the depletion of several genes. 12. 0.8% Agarose gel: add 4 g of agarose and 300 ml of 1× TBE in a 500-ml glass bottle.

Materials

Cloning of Cre-recombinase or Reporter Genes into

Heat the mix in the microwave oven until the agarose is dissolved and make up the volume to 500 ml with 1× TBE. Add 20 µl of 10 mg/ml Ethidium Bromide solution and store in incubator set at 55°C for not more than two weeks. Prepare the gel tray and comb of desired size, pour the agarose and let polymerize at room temperature for at least 15 min. 18. LB-agar plates containing 100 µg/ml of Ampicillin 19. LB medium without antibiotic used for recovery of bacteria 20. LB medium with 100 µg/ml of ampicillin 21. 14-ml round-bottom tubes 22. Bacterial incubator with shaking set at 37°C.

Lentivirus Production

1. X-Lenti 293T (Takara) (see Note 2).

2.

Cell culture incubator at 37°C with 5% CO2. 21. B27 medium: add 2 ml of 50× B27 solution and 1 ml of Glutamax TM supplement to 97 ml of neurobasal medium-PS. Prepare fresh for each experiment.

22. 2× B27 medium: add 2 ml of 50× B27 solution and 1 ml of Glutamax TM to 47 ml of neurobasal-PS medium. Prepare fresh for each experiment.

Genotyping

1. 2% Agarose: add 10 g of agarose and 300 ml of 1× TBE in a 500-ml glass bottle. Heat the mix in the microwave oven until the agarose is dissolved and make up the volume to 500 ml with 1× TBE. Add 20 µl of 10 mg/ml Ethidium Bromide solution and store in incubator set at 55°C for not more than two weeks. Prepare the gel tray and comb of desired size, pour the agarose and let polymerize at room temperature for at least 15 min.

2. 100 bp DNA Ladder.

3. 10 mg/ml Proteinase K: Dissolve 100 mg of proteinase K in 10 ml of autoclaved distilled water. Aliquot and store at -20°C indefinitely.

4. 1 M Tris-HCl pH 8: dissolve 12.1 g of Tris in 100 ml autoclaved distilled water and adjust to pH 8 with HCl. Filter sterilize and store at room temperature.

5. 5 M NaCl: dissolve 29.2 g of NaCl in 100 ml autoclaved distilled water. Filter sterilize and store at room temperature. 6. 0.5 M EDTA pH 8: dissolve 14.6 g of EDTA in 100 ml autoclaved distilled water and adjust to pH 8 with KOH. Filter sterilize and store at room temperature.

7. Tail lysis buffer: 0.1 M Tris-HCl pH 8, 0.2 M NaCl, 5 mM EDTA and 0.4% SDS. Mix 50 ml of 1 M Tris-HCl pH 8, 20 ml of 5 M NaCl, 5 ml of 0.5 M EDTA pH 8 and 10 ml of 20% SDS and complete the volume to 500 ml with distilled water.

8. Specific primers. 9. For CCP1, primer mix 1: mix primers (5'-TTAAGCAGTGGCTGCCGGAGTGC-3') and (5'-GTCTACAGCCACGTGCTCAGCAAAGG-3') at 1:1 and primer mix 2: Mix primers (5'-GGTCATCTTGTAACAGTAGGGAAATCAAGCC-3') and (5'-GGAAAGCATAGCATCATGTTAAAACTCCCTCC-3') at 1:1. 10. For CCP6, primer mix 1: mix primers (5'-GAATGGCAATGAGATCACCACTCTCCAGC -3') and (5'-CTGTTGGGTGTCTGAGGCAAACACTTCC -3') at 1:1 and primer mix 2: Mix primers (5'-AGGTGTTCAGTGTAAAAGGAATCTGGTCCC -3') and (5'-CTGTTGGGTGTCTGAGGCAAACACTTCC -3') at 1:1.

11. GoTaq ® DNA polymerase (Promega), use according to manufacturer's instructions.

12. Electrophoresis cell: Mini-PROTEAN ® cell with a PowerPac™power supply (Bio-Rad).

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

1. 5× Laemmli sample buffer: 400 mM Tris-HCl pH 6.8, 450 mM DTT, 10% (wt/vol) SDS and 50% (vol/vol) glycerol. Preheat 16 ml of 1 M Tris-HCl pH 6.8 to app. 40°C, add 2.6 g of DTT and 4 g of SDS. Stir the solution gently using a magnetic stirrer and add 20 ml of 100% glycerol. Continue stirring until the solution is homogenous and add a pinch of Bromophenol blue to reach the desired intensity of colour. Aliquot and store at -20°C indefinitely.

2. Acrylamide/bis-acrylamide stock solution: 40% Acrylamide supplemented with 1.04% (wt/vol) Bis N,N'-Methylene-Bis-Acrylamide. Store at 4°C for up to 6 months. 13. TBST containing 5% (wt/vol) fat-free dry milk: Add 0.5 g of milk to 10 ml TBST.

14. Antibodies: 12G10, anti-a-tubulin antibody (used at 1/500 (vol/vol), developed by J.

Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa). PolyE, anti-polyglutamylated tubulin antibody (used at 1/20,000 (vol/vol); AdipoGen). Anti-GFP antibody (used at 1/5,000 (vol/vol); Acris Antibodies).

15. Clarity™ Western ECL Substrate (Biorad) [START_REF] Silva | Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis[END_REF]. Western blot developer or imager

Methods

Here, we describe a method to obtain primary neurons in which we can induce the knockout of tubulin-modifying enzymes thus obtaining strong alterations in the levels of specific tubulin PTMs. As an example, we describe the protocol to generate hyperglutamylation in primary hippocampal neurons, which can be used to study the role of polyglutamylation in several neuronal functions, such as axonal outgrowth and branching, neuronal connectivity, or axonal transport.

Briefly, we culture hippocampal neurons from Ccp1 flox/flox Ccp6 flox/flox E17.5 (embryonic day 17.5) mouse embryos and transduce them either with a GFP-or GFP-2A-cre-expressing lentivirus at DIV 0 (days in vitro). The use of lentivirus-mediated gene transfer allows a nearly complete transduction of all cells in the culture dish, and appears to not exert additional stress on the neurons, as we observe no neuronal death.

Cre-recombinase was cloned into the pTRIP vector under the CMV or the CMV-enhanced chicken beta-actin (CAG) promoter using one step sequence and ligation independent cloning (SLIC) method [START_REF] Jeong | One-step sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies[END_REF]. Considering the possibility that direct GFP-tagging of Cre protein might affect its recombinase activity, we cloned Cre behind a self-cleavable 2A peptide sequence [START_REF] Kim | High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice[END_REF] present in frame with the GFP protein sequence in the vector (Fig. 1a). In cells, the crerecombinase is cleaved from GFP, which we confirmed by immunoblotting (Fig. 2b).

Transduction of Ccp1 flox/flox Ccp6 flox/flox neurons with GFP-2A-cre resulted in 'defloxing' of both alleles of the two genes (Fig. 4a), and a concomitant increase in tubulin polyglutamylation (Fig. 4b). In parallel, wild type neurons were transduced with the same GFP-and GFP-cre viruses to confirm that the cre-recombinase alone has no effect on polyglutamylation levels (Fig. 4c).

Thus, obtained neurons can be used to determine the impact of increased polyglutamylation on different neuronal processes including neuronal differentiation and intracellular transport, by staining organelles using commercially available dyes such as Mitotracker and Lysotracker. Furthermore, this protocol combined with lentivirus-based expression of reporter genes can extend the applications of this method to study the trafficking of other vesicles such as endosomes (Rab5, Rab7 and LAMP1), BDNF vesicles (BDNF), autophagosomes (LC3), or synaptic vesicles (Synaptotagmin, Synaptophysin). Alternatively, the system can be used to study microtubule dynamics by the use of end-binding protein-EB3 fused to GFP.

Cloning of Cre-Recombinase and Reporter Genes into a Lentiviral Vector

Cloning of Cre-recombinase into the pTRIP vector -generation of the pTRIP-

CMV-GFP-2A-Cre plasmid (Fig. 1).

1. Linearize the pTRIP vector by restriction digestion with XhoI (Fig. 1a). Digest 3 µg of plasmid DNA for ~2.5 h with 0.5 µl of XhoI enzyme (20 U/µl) in a 50 µl reaction.

Run the whole sample on a 0.8% agarose gel until the dye front runs out. (See Note 8)

2. Purify the digested vector from the gel using the gel purification kit. Elute the vector DNA in 50 µl of elution buffer.

3. Amplify the cre-recombinase gene using the primers containing at least 15 bp of homology to the ends of the vector. In a PCR tube, mix 25 µl of 2× KAPA ready mix, 0.2 µl of each primer (100 µM), PCR substrate and make up the volume to 50 µl with DNase/RNase-Free distilled water and perform the PCR reaction. Use the following PCR conditions: an initial step of denaturation at 95°C for 3 min; 15 cycles of amplification (95°C for 30 sec, annealing of primers at 65°C for 30 sec, and extension at 72°C for ~1.5 min). (See Note 9). 4. Run the whole sample on a 0.8% agarose gel and purify the PCR product from the gel using the gel purification kit. Elute the PCR product in 40 µl of elution buffer. 5. Mix 7 µl of insert or elution buffer (negative control), 2 µl of digested vector and 1 µl of buffer 2.1 in to a 1.5-ml tube, centrifuge briefly and place on ice (Fig. 1b).

6. Add 0.3 µl of T4 DNA polymerase (3 U/µl) to each tube and mix by firmly tapping the tube (Fig. 1b).

7. Centrifuge briefly and leave the reaction mix at room temperature for 2.5 min to generate 5' over-hangs (Fig. 1b). (See Note 10).

After the incubation, immediately transfer the tubes on ice to stop the activity of T4

DNA polymerase and leave it for ~5 min to facilitate the annealing of single-stranded over-hangs (Fig. 1b). 9. Add 100 µl of chemically competent E. coli to the DNA reaction and mix by gentle tapping at the bottom of the tube. 10. Leave the E. coli with the reaction mix on ice for 30 min, provide heat shock at 42°C for 45 s, bring back the tube on ice for 10 min. Add 1 ml of LB medium without antibiotics and let bacteria recover at 37°C for at least 1 h. 11. After the incubation, pellet the E. coli cells at room temperature, 1,000 RCF for 3 min, resuspend in ~100 µl of LB medium, plate the entire cell suspension on a LB-agarampicillin plate and incubate at 37°C for 16 h. 12. Next day, inoculate 5 to 10 colonies, each in a 14-ml round bottom tube with ~ 5 ml of LB medium containing ampicillin. (See Note 11).

13. Extract the plasmid DNA and test the clones by restriction digestion with appropriate enzymes. Finally, verify the positive clones by sequencing.

Cloning of reporter genes into pTRIP lentivectors -generation of the pTRIP-

CMV-reporter-GFP-2A and pTRIP-CMV-reporter-GFP-2A-Cre plasmids.

1. Linearize pTRIP-CMV-GFP-2A and pTRIP-CMV-GFP-2A-Cre lentivectors by restriction digestion with NheI (Fig. 1a). Digest 3 µg of plasmid DNA for ~2.5 h with 0.5 µl of NheI enzyme (20 U/µl) in a 50-µl reaction. Gel-purify the digested vector in 50 µl of elution buffer. (See Note 12).

2. Amplify reporter gene using the primers containing at least 15 bp of homology to the ends of the vector. In a PCR tube, mix 25 µl of 2× KAPA ready mix, 0.2 µl of each primer (100 µM), PCR substrate and make up the volume to 50 µl with DNase/RNase-Free distilled water.

3. Then follow the same procedure as in section 3.1.1, from step 3 onwards.

Lentivirus Production and Transduction (Fig. 2a) (See Note 13)

Grow X-lenti 293T cells in a 75 cm 2 flask to near confluence. It is critical not to let the cells become over-confluent as it will affect the efficiency of virus production. From a confluent 75-cm 2 flask, ~10×10 6 cells can be obtained. Pre-warm Trypsin, PBS, and DMEM complete culture medium to 37°C.

Day -1: Seeding cells for transfection

1. Remove the medium from the 75 cm 2 flask and gently wash the cells with 5 ml of prewarmed PBS to remove any excess medium left in the flask.

2. Add 1 ml of Trypsin to the flask and leave in the incubator for not more than 1 min. Collect all the trypsinized cells in culture medium.

Pellet down the cells by centrifugation at 150 RCF for 2 min.

4. Re-suspend the cells gently in 1 ml of complete culture medium. It is important to dissociate the cells adequately, as leaving cell clumps will reduce the transfection efficiency in later steps.

5. Count and dilute the cells to 0.15×10 6 cells per ml in complete culture medium.

6. Add 3 ml of the cell suspension (0.45×10 6 cells) per well of a 6-well plate and leave the plate in the cell culture incubator (37°C, 5% CO2).

Day 0: Transfection of plasmid DNA

Pre-warm the DMEM complete culture medium and plain DMEM to 37°C. The following protocol gives volumes for transfection of one well of a 6-well plate for each lentiviral vector, GFP and GFP-2A-Cre.

1. Mix 1.6 µg of lentiviral plasmid of interest with 0.4 µg of pCMV-VSVG and 1 µg of psPAX2 in a 1.5-ml tube. 6. During the incubation time, replace the medium of the cells with 2 ml of fresh, prewarmed complete culture medium.

Mix the

Transduction of neurons

In this protocol we assume that the volume of the virus added to the neurons constitutes 50% of the total volume of the culture medium. As the virus-containing-medium (Neurobasal) does not contain the supplements necessary for neurons to grow, it is essential to add twice the final amount of these supplements to the medium (2× B27 medium) used to complement the virus-containing medium volume.

1. Prepare fresh (see chapter 3.2), or thaw the required amount of the desired lentiviruses (for example GFP-and GFP-2A-Cre encoding lentiviruses) under the hood.

2. In the meantime, remove the plating medium and add 0.5 volume of 2× B27 medium to the neurons. For example, for a 35-mm glass-bottom dish add 1 ml of 2× B27 medium.

3. Once the virus is completely thawed, add 0.5 volume of the lentivirus-containing medium (for a 35-mm glass bottom dish, add 1 ml of lentivirus). For untransduced samples, add Neurobasal-PS medium. Rapidly return the cells to the cell culture incubator.

Genotyping

Prior to the analysis of the transduced neurons, confirm the genotype of neurons in order to verify the activity of the cre-recombinase.

1. Add 100 µl of the lysis buffer containing proteinase K to each well of 12-well dish and lyse the neurons using a p200 micropipette.

2. Incubate the lysate at 55°C for at least 4 h or overnight.

3. Boil the samples at 95°C for 10 min. 4. In order to determine the genotype of CCP1 and CCP6, perform PCR reactions using primer mix 1 and primer mix 2 for both genes. Use the following PCR conditions: an initial step of denaturation at 96°C for 5 min; 35 cycles of amplification (96°C for 30 sec, annealing of primers at 63.5°C for 30 sec, and extension at 72°C for ~30 sec).

6. Run 10 µl of the PCR product on the 2% agarose gel. (See Note 27).

7. For CCP1, primer mix 1 amplifies ~400 bp fragment in the flox allele (and no product in the knockout allele) and the primer mix 2, ~600 bp fragment in the knockout allele (and no product in the flox allele). For CCP6, primer mix 1 amplifies ~440 bp fragment in the flox allele (and no product in the knockout allele) and the primer mix 2, ~800 bp fragment in the knockout allele (and no product in the flox allele) (Fig. 4a).

Note, that the primer mix 1 still amplifies a faint band in both CCP1 and CCP6, suggesting a residual presence of flox alleles, which could come from untransduced cells.

SDS-polyacrylamide gel electrophoresis and immunoblotting

Prior to the analysis of the transduced neurons, collect the samples to later verify the effect of the depletion of CCP1 and CCP6 on the levels of tubulin polyglutamylation in transduced neurons by immunoblot (Fig. 4b).

1. Add 200 µl of the Laemmli sample buffer to each well of 6-well dish and lyse the neurons using a p200 micropipette. (See Note 28).

2. Boil the samples at 95°C for 5 min and spin down briefly.

3. Set up the gel casting apparatus according to manufacturer's description. For one 1-mm-thick 10% acrylamide gel, mix 1.25 ml of acrylamide/bis solution, 1.25 ml of 4× resolving buffer and 2.5 ml of distilled water in a 15-ml tube. Add 40 µl of 10% APS and 5 µl of TEMED, mix gently by inverting the tube and immediately pipette the solution into the preassembled casting apparatus. Add a small layer of distilled water (~300 µl) on the gel solution and leave it undisturbed for at least 30 min.

4. Remove the overlaid water layer by inverting the gel and drain off the remaining water droplets with a tissue paper.

5. Prepare the stacking gel: for one 1 mm-thick resolving gel, mix 175 µl of acrylamide/bis solution, 406 µl of 4× stacking buffer and 1044 µl of distilled water. Add 10 µl of 10% APS and 4 µl of TEMED, mix gently by inverting the tube and immediately pipette the solution onto the polymerized resolving gel. Carefully insert a comb into the stacking gel and allow it to polymerize for 30 min.

6. Assemble the electrophoresis tank and fill the chamber with the gel running buffer.

Remove the comb and wash the wells with the buffer. 7. Boil the samples at 95°C for 5 min, spin down briefly, load the samples on the gel and separate the proteins at 200 V until the dye front runs out from the gel. (See Note 29).

8. Transfer the separated proteins from the gel to a nitrocellulose membrane using the Biorad Trans-Blot ® Turbo system, according to manufacturer's instructions. 9. Block the membrane in TBST containing 5% skimmed milk for 1 h, with gentle shaking, at room temperature. 10. Incubate the membrane with primary antibodies (12G10, PolyE or anti-GFP) diluted in TBST containing 2.5% skimmed milk for 1 h, with gentle shaking, at room temperature.

11. Wash the membrane 3×5 min in TBST with vigorous shaking.

12. Incubate the membrane with secondary, HRP-conjugated antibodies (anti-mouse for 12G10; anti-rabbit for poly E and anti-GFP) diluted in TBST buffer for 1 h, with gentle shaking, at room temperature.

13. Wash the membrane 3×5 min in TBST with vigorous shaking.

14. Reveal the chemiluminescence signal on the membrane by incubating it with the ECL solution according to manufacturer's instructions and using the available equipment (either chemical or digital imager) 4.

Notes

1. The primer sequence written in lower case shares homology with the ends of the target vector, pTRIP-CMV-GFP-2A 2. Always use cells which have been in culture for no more than 5-8 passages, as using cells after multiple passages may reduce the viral titre. It is very important to ensure that Xlenti 293T cells are mycoplasma free, as mycoplasma contamination can affect the virus production efficiency.

3. We have used other transfection reagents such as jetPEI for virus production and found that the virus titre produced is lower than when using TransIT-293.

4. We used pTRIP vector with CAG and CMV promoters for the experiments. In our experience, there is no difference in their expression levels in neurons at young age.

However, if transduction to be performed at a differentiated stage, we suggest to use vectors with CAG promoter, as it expressed better than CMV promoter in mature neurons.

5. The choice of medium used for virus-production depends on the medium preferred by the cells you intend to transduce. In this case, we used Neurobasal medium as our target cells were neurons.

6. If there is a need to separate alpha-and beta-tubulins on the SDS-PAGE, we recommend using a different gel composition [START_REF] Magiera | Investigating tubulin posttranslational modifications with specific antibodies[END_REF].

7. As Tween 20 is highly viscous, add it to the TBS solution using a cut p1000 pipette tip and rinsing the tip by repeated pipetting 8. It is crucial to run the gel for a sufficiently long time, as it will aid in separating the cut from the uncut vector, reducing the contamination with uncut vector during purification of digested vector. 9. Extension time needs to be adjusted based on the length of the amplicon and speed of the polymerase. In our case, we consider that polymerase requires ~1 min to amplify ~1 kb of DNA template 10. The timing of the reaction mix is to be strictly followed. Leaving the reaction mix for less or more time will reduce the efficiency of SLIC.

11. Usually, when the SLIC reaction is efficient, we get ~80 colonies in the positive plate and ~5 colonies in negative control plate. If the SLIC efficiency is poor, you will get less colonies in the positive plate. If the vector is not properly digested, you will get many colonies in the negative control plate.

12. NheI is selected when the reporter gene is to be C-terminally GFP-tagged. Use BsrGI enzyme when the reporter gene is to be N-terminally GFP-tagged. [START_REF] Grau | Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration[END_REF]. Rules and regulations regarding working with lentiviral particles are specific to each country. Please enquire about and follow the rules that apply in your country.

14. Always add the diluted TransIT-293 into DNA mix, and not the other way around.

15. Usually we add 3 ml of virus collection medium per well of a 6-well plate. It is possible to obtain a more concentrated virus by adding a lower volume of medium. The minimal volume possible is 1.5 ml. [START_REF] Silva | Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis[END_REF]. Avoid exceeding this time, as it might reduce the competence of produced lentivirus. 17. Do not use 0.2 µm filter, as it might damage the lentivirus. 22. Success of primary neuron culture depends on the time of dissection (from sacrificing the dam to collecting all the hippocampi) and should take no longer than 90 min.

23. Be careful as the hippocampi tend to be aspirated by the suction created by pipetting. If it is difficult to remove the trypsin without the hippocampi, proceed immediately to washes.

The tendency of hippocampi to be aspirated with the pipette suction reduces after washes with plating medium. 26. For live-cell imaging and immunostaining, it is important to plate neurons at a lowdensity. Neurons seeded at high-density will have overlapping axons and dendrites, making it difficult to use them for analysis. [START_REF] Kim | High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice[END_REF]. Usually, 10 µl of PCR product is sufficient to visualize the amplification. If there is an unexpected problem with the gel, make a fresh gel and run another 10 µl from the remaining PCR product.

28. Depending on the day of the experiment, the sample might need to be further diluted before immunoblotting in order to not overload the gel with protein.

29. When analysing the differences in levels of tubulin modifications, we strongly recommend to load low amounts of protein on the gel [START_REF] Magiera | Investigating tubulin posttranslational modifications with specific antibodies[END_REF].

Lentiviral Vector 1 .

 1 pTRIP vector with CMV promoter, pTRIP-CMV-GFP-2A (modified from Addgene #102611) 2. Template for PCR reaction (brain cDNA or plasmid containing gene of interest) 3. KAPA HiFi HotStart 2× ReadyMix (Kapa Biosystems) 4. Specific primers NLS-cre-FS-2A: 5'-tccactagtgtcgacATGCCCAAGAAGAAGAGGAAGGTG-3' NLS-cre-FS-2A: 5'-catgtttttctaggtTAATCGCCATCTTCCAGCAGGC-3Tris-Borate-EDTA (TBE) buffer: add 100 ml of 10× TBE to 900 ml of distilled water.

16 .

 16 Chemically competent E. Coli strain Dh5alpha 17. Bacterial incubator at 37°C.

3 . 3 .

 33 Tissue culture treated 75-cm 2 flasks. 4. Phosphate-buffered saline (PBS). 5. Trypsin. 6. Plain medium: Dulbecco`s Modified Eagle Media (DMEM) without any supplements. 7. Complete culture medium: Supplement DMEM with a final concentration of 10% FBS (vol/vol), 200 mM L-Glutamine, and 1× Penicillin-Streptomycin. Store at 4°C. 8. Tissue culture treated 6-well plates. 9. Tissue culture treated 24-well plates. 10. Transfection reagent: TransIT-293 (Mirus Bio LLC) (see Note 3). 11. Expression and packaging plasmids: pTRIP lentiviral vector containing gene of interest under the CMV or CMV-enhanced chicken beta-actin (CAG) promoter and packaging plasmids including psPAX2 (a gift from D. Trono, Addgene plasmid #12260) and pCMV-VSVG (a gift from B. Weinberg, Addgene plasmid #8454). (See Note 4). 12. Virus collection medium: Neurobasal medium-PS: supplement Neurobasal medium with a final concentration of 1× Penicillin-Streptomycin. Store at 4°C. (See Note 5). 13. 15-ml and 50-ml screw-cap tubes 14. 0.45 µm filters and 20-ml syringes 15. 1.5-and 2-ml tubes 2.3 Mouse Primary Hippocampal Neuron Cultures and Lentiviral Transduction 1. DNase/RNase-Free Distilled Water 2. 1 mg/ml poly-D-Lysine: add 50 ml of DNase/RNase-Free distilled water to 50 mg of poly-D-Lysine bottle. Aliquot by 5 ml or 10 ml in sterile 15-ml tubes and store at -20°C. Autoclaved distilled water 4. Tissue culture treated 75-cm 2 flasks 5. Dissection medium: add 10 ml of 10× HBSS and 1 ml of 1 M HEPES solution to 89 ml of DNase/RNase-Free distilled water. 6. Dissection tools 7. Tissue culture-treated 100-mm dishes 8. Tissue culture-treated 60-mm dishes 9. 15-ml screw-cap tubes 10. 10× Trypsin (2.5%), no phenol red. Aliquot by 500 µl in 1.5-ml tubes and store at -20°C. Avoid repeated freeze-thaw cycles. 11. Plating medium: add 5 ml of filter sterilized heat inactivated horse serum, 1.3 ml of glucose and 1 ml of 100× Penicillin-Streptomycin to 93.7 ml of Minimum Essential Medium. Prepare fresh before use. 12. 70-µm cell strainers 13. KOVA™ Glasstic™ Slide 10 with Grids (Fisher Scientific) 14. 35-mm glass bottom dishes 15. Tissue culture treated 6-well plates 16. Tissue culture treated 24-well plates 17. 12-mm, No.1.5H, round cover slips 18. Tissue culture treated 12-well plates 19. B27 supplement (Thermo Fisher). Aliquot by 1 ml in 1.5-ml tubes and store at -20°C. Avoid repeated cycles of freeze-thawing. 20. Neurobasal-PS: supplement Neurobasal medium with a final concentration of 1X Penicillin-Streptomycin. Store at 4°C.

3 .

 3 4× resolving gel buffer: 1.5 M Tris-HCl pH 8.8 and 0.4% SDS. Dissolve 36.3 g of Tris in autoclaved distilled water and adjust to pH 8.8 with HCl for a final volume of 396 ml. Then, add 4 ml of 20% SDS stock solution. Filter-sterilize and store at room temperature indefinitely. (See Note 6).

4 .

 4 4× stacking gel buffer: 0.5 M Tris-HCl pH 6.8 and 0.4% SDS. Dissolve 12.1 g of Tris in autoclaved distilled water and adjust to pH 6.8 with HCl for a final volume of 196 ml. Then, add 4 ml of 20% SDS stock solution. Filter-sterilize and store at room temperature indefinitely. 5. N, N, N′, N′-Tetramethylethylenediamine (TEMED).6. 10% Ammonium persulfate (APS): Dissolve 1 g of APS in 10 ml of autoclaved distilled water. Aliquot and store at -20°C indefinitely. 7. SDS-PAGE electrophoresis equipment (Bio-Rad) 8. Electrophoresis cell: Mini-PROTEAN ® cell with a PowerPac™power supply 9. Gel running buffer: 50 mM Tris-HCl, 384 mM glycine and 0.1% SDS. Dissolve 6 g of Tris and 28.75 g of glycine in autoclaved distilled water for a final volume of 995 ml. Then, add 5 ml of 20% SDS stock solution. 10. Trans-Blot ® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad) 11. Trans-Blot ® Turbo™ Transfer System (Biorad) 12. Tris-buffered saline -Tween 20 (TBST) solution: 20 mM Tris-HCl pH 7.4, 140 mM NaCl and 0.1% Tween 20. Dissolve 2.42 g of Tris and 8.18 g of NaCl in autoclaved distilled water and adjust to pH 7.4 with HCl for a final volume of 999 ml. Add 1 ml of Tween 20 to the buffer and mix well. (See Note 7).

5 .

 5 TransIT-293 transfection reagent by gently inverting the tube. Add 8 µl of TransIT-293 to 200 µl of plain DMEM medium in a 1.5-ml tube. 3. Mix the diluted TransIT-293 well by vortexing for ~10 s and spin shortly to collect all the liquid in the bottom of the tube. 4. Add 200 µl of the diluted TransIT-293 reagent to 3 µg of plasmid DNA mix. (See Note 14). Mix the tube gently by inverting and leave under the hood for 15-20 min to allow the formation of TransIT-293-DNA transfection complexes.

5 .

 5 Set up separate PCR reactions for each primer mix (25 µl): Add 18 µl of water, 5 µl of 5× buffer, 0.5 µl of 10 mM dNTPs, 0.5 µl of GoTaq ® DNA polymerase (1 U/µl), 0.1 µl of primer mix and 1 µl of DNA sample.

18 .

 18 As viruses are very sensitive to mechanical stress, do not accelerate thawing by pipetting or shaking. 19. As a general rule, transduction of primary cell cultures is difficult compared to cell lines. Therefore, the intensity of GFP-fluorescence in U-2 OS cells and neurons might be different. If the GFP intensities are very different, then we suggest to use wild type neurons for testing and determining the amount of virus to be added. 20. For cleaning coverslips, place a maximum of 200 coverslips in a 100-ml glass bottle. Wash them by shaking for at least 30 min in 3 M HCl, 2×10 min in distilled water, 3×10 min in 95% EtOH and 2×10 min in distilled water. Let the coverslips dry under the hood or in an oven. 21. Proper coating of the surface with poly-D-Lysine is crucial for attachment and development of the neurons. If the dishes are not coated properly, it will cause the neurons to clump or not adhere well.

24 .

 24 Do not exceed the number of trituration passages through the Pasteur pipettes, even if pieces of tissue are still visible at the end of the procedure. 25. Some labs use 40 µm cell strainers to pass the cell suspension. In our hands, 70-µm cell strainers worked best without affecting the survival of neurons.

Fig. 1 .

 1 Fig. 1. Strategy used to clone the cre-recombinase and reporter genes into the pTRIP lentiviral vector. (a) Vector map of the pTRIP-CMV-EGFP-2A lentiviral plasmid. To allow the cleavage of cre recombinase and GFP, cre was inserted into the XhoI site. For further insertion of additional reporter genes, the backbone can be opened with NheI to generate a Cterminally GFP-tagged fusion protein, or with BsrGI to generate a N-terminally GFP-tagged fusion protein. (b) Flowchart of the sequence-and ligation-independent cloning (SLIC)method (adapted from[START_REF] Jeong | One-step sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies[END_REF]).

Fig. 2 .

 2 Fig. 2. Production and functional analysis of the lentiviral particles. (a) Flowchart of the production of lentiviral particles. X-lenti 293T cells are transfected with the envelope vector (pCMV-VSV-G), packaging vector (psPAX2) and the backbone pTRIP vector containing the gene of interest. On the day following the transfection, the medium is replaced by the desired virus-collection medium, which is collected 24-36 h later, filtered and aliquoted. The medium can either be directly used to transduce cells, or frozen for later use. Note that the medium changes colour to more orange during virus production. (b) Immunoblots showing the efficiency of the cleavage of the P2A site between the GFP and the cre recombinase. Extracts from U-2OS cells transduced with lentivirus encoding either GFP, or GFP-2A-crerecombinase are immunoblotted with anti-GFP antibody. In both cases, only a single band of ~28 kDa, corresponding to GFP, is detected. The absence of a higher-molecular-weight band, corresponding to the fusion protein GFP-cre-recombinase, demonstrates that all GFP-cre fusion protein was cut. Total a-tubulin (detected by the 12G10 antibody) is used as a loading control.

Fig. 3 .

 3 Fig. 3. Mouse primary hippocampal neuron culture and the experimental design. (a) Representative pictures of successive steps of hippocampus dissection from an E17.5 embryonic brain. Scale bar: 2 mm. (b) Representative images of unpolished, fire-polished and reduced-diameter Pasteur pipettes used to dissociate hippocampi. Scale bar: 2 mm. (c) Schematic representation of the culture dishes needed for one experiment including nontransduced, GFP-and GFP-2A-cre-transduced neurons.

Fig. 4 .

 4 Fig. 4. Analysis of efficiency of the cre-mediated recombination in primary hippocampal neurons. (a) An agarose gel showing the genotypes of Ccp1 flox/flox Ccp6 flox/flox neurons transduced with GFP-and GFP-2A-cre viruses. Note that in the presence of GFP alone, only flox alleles are detected, while with GFP and cre recombinase the major band comes from the

Acknowledgments

This work was supported by the ANR-10-IDEX-0001-02, the LabEx CelTisPhyBio ANR-11-LBX-0038. CJ is supported by Institut Curie, the French National Research Agency (ANR) awards ANR-12-BSV2-0007 and ANR-17-CE13-0021, the Institut National du Cancer (INCA) grant 2014-PL BIO-11-ICR-1, and the Fondation pour la Recherche Medicale (FRM) grant DEQ20170336756. MMM is supported by the EMBO short-term fellowship ASTF 148-2015 and by the Fondation Vaincre Alzheimer grant FR-16055p, and SB by the FRM grant FDT201805005465. We thank C. Alberti, E. Belloir, F. Bertrand, V. Dangles-Marie, I. Grandjean, C. Caspersen, H. Hermange, A. Thadal, G. Buhagiar, C. Serieyssol, S. Gadadhar, M. Sittewelle, (Institut Curie) for technical assistance. We are grateful to M.-N. Soler, C. Lovo and L. Besse from the PICT-IBiSA@Orsay Imaging Facility of the Institut Curie supported by the ANR through the "Investment for the future" program (France-BioImaging, ANR-10-INSB-04), and to N. Manel (Institut Curie, Paris) for material and advice for the lentivirus production. We would like to thank F. Del Bene, V. Marthiens (Institut Curie) and C. González-Billault (University of Chile, Santiago, Chile) for instructive discussions and advice.


D Payandi-Rolland
email: dahedrey.payandi-rolland@get.omp.eu

L S Shirokova

M Tesfa

P Bénézeth

A G Lim

D Kuzmina

J Karlsson

R Giesler

O S Pokrovsky

Dissolved organic matter biodegradation along a hydrological continuum in permafrost peatlands

Keywords: river, stream, thermokarst lake, supra-permafrost water, carbon dioxide emission, low molecular weight organic acids

We compared surface waters of two hydrological continuums (HC) in northern Sweden and western Siberia

• Large spatial heterogeneity of dissolved organic carbon concentration and biodegradability along HC.

• Along the HC from supra-permafrost waters, fens, and ponds to lakes, streams and rivers, the DOC removal rate decreased and the BDOC increased.

• BDOC ranged from 0 to 20% and could at the most account for 10% of CO2 emissions from surface waters of these permafrost peatlands

Introduction

The carbon (C) released from thawing permafrost is biotically and abiotically processed in aquatic systems (Abbott et al., 2016;[START_REF] Cory | Interactions between sunlight and microorganisms influence dissolved organic matter degradation along the aquatic continuum[END_REF][START_REF] Mann | Utilization of ancient permafrost carbon in headwaters of Arctic fluvial networks[END_REF]. However, the degree and intensity of this processing are strongly variable both in space (local to global scale) and time (day/night to season) and not explicitly accounted for in global modelling of permafrost-carbon-climate feedback. The spatial variability includes the various types of water bodies of those regions, all belonging to the hydrological continuum (Figure 1). Globally, the concept of 'hydrological continuum' [START_REF] Creed | The river as a chemostat: fresh perspectives on dissolved organic matter flowing down the river continuum[END_REF][START_REF] Palmer | Sporadic hotspots for physicochemical retention of aquatic organic carbon: from peatland headwater source to sea[END_REF] includes the movement of water from sources (soil and supra-permafrost waters) to intermediate reservoirs (lakes and streams) and finally into terminal reservoirs (large rivers and their estuaries). In Arctic regions, the hydrological continuum (HC) is highly dependent on the maturation cycle of water bodies. All steps of HC are especially important as they can influence the biochemical properties, photo- [START_REF] Panneer Selvam | Photo-reactivity of dissolved organic carbon in the freshwater continuum[END_REF] and bio-degradability [START_REF] Liu | Disentangling the Effects of Climate, Vegetation, Soil and Related Substrate Properties on the Biodegradability of Permafrost-Derived Dissolved Organic Carbon[END_REF][START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF] of dissolved organic matter (DOM). Since the transport of thawed permafrost organic C can spatially relocate the emissions of greenhouses gases [START_REF] Vonk | Permafrost-carbon complexities[END_REF], the heterogeneity of waterbodies observed along a hydrological continuum is a key point to understand the C cycle in these regions.

Dissolved organic carbon (DOC) is the main form of terrestrial organic carbon (OC) exported to soil solution, rivers, wetlands and lakes [START_REF] Chapin | Reconciling Carbon-cycle Concepts, Terminology, and Methods[END_REF][START_REF] Cole | Plumbing the Global Carbon Cycle: Integrating Inland Waters into the Terrestrial Carbon Budget[END_REF]. This is especially true in the case of discontinuous permafrost regions, where the hydrologic flow paths have longer residence times and allow a greater degree of DOC processing during lateral transport (Olefeldt andRoulet, 2014, 2012;[START_REF] Tang | Drivers of dissolved organic carbon export in a subarctic catchment: Importance of microbial decomposition, sorption-desorption, peatland and lateral flow[END_REF][START_REF] Walvoord | Increased groundwater to stream discharge from permafrost thawing in the Yukon River basin: Potential impacts on lateral export of carbon and nitrogen[END_REF]. For these reasons, coupled aquatic-soil studies of organic matter (OM) in continuous-discontinuous permafrost regions are needed [START_REF] Vonk | Integrating hydrology and biogeochemistry across frozen landscapes[END_REF]. The importance of surface waters from frozen peatlands in DOC processing and CO2 emissions motivated numerous studies of aquatic DOM [START_REF] Hulatt | Bioavailability and radiocarbon age of fluvial dissolved organic matter (DOM) from a northern peatland-dominated catchment: effect of landuse change[END_REF][START_REF] Manasypov | Seasonal dynamics of organic carbon and metals in thermokarst lakes from the discontinuous permafrost zone of western Siberia[END_REF][START_REF] Mann | Utilization of ancient permafrost carbon in headwaters of Arctic fluvial networks[END_REF][START_REF] Mann | Controls on the composition and lability of dissolved organic matter in Siberia's Kolyma River basin[END_REF][START_REF] Peura | Ontogenic succession of thermokarst thaw ponds is linked to dissolved organic matter quality and microbial degradation potential[END_REF][START_REF] Pokrovsky | Organic and organomineral colloids in discontinuous permafrost zone[END_REF][START_REF] Shirokova | Humic surface waters of frozen peat bogs (permafrost zone) are highly resistant to bio-and photodegradation[END_REF]. The latter study in NE European tundra peatlands demonstrated weak biodegradation of DOM along a hydrological continuum from peatland subsidence to a large river (Pechora) and suggested that CO2 emission from surface waters could be explained either by sediment respiration or by fast processing of fresh suprapermafrost flow delivered from peat pore water and thawing soil ice. As such, to quantify overall DOM biodegradation potential in Arctic peatland waters, it is necessary to study the full continuum of surface fluids, from the soil and supra-permafrost waters to large lakes or river systems using universal techniques over large geographic coverage [START_REF] Ma | Dissolved organic carbon in permafrost regions: A review[END_REF].

This study aimed to quantify the magnitude and controlling factors of DOM biodegradation potential along a hydrological continuum from soil water to medium-sized rivers and lakes located in a discontinuous permafrost area in western Siberia and northern Sweden. Strong seasonal variability in the high latitude C cycle is well known [START_REF] Gao | Riverine dissolved organic carbon and its optical properties in a permafrost region of the Upper Heihe River basin in the Northern Tibetan Plateau[END_REF][START_REF] Li | Distribution, seasonality, and fluxes of dissolved organic matter in the Pearl River (Zhujiang) estuary, China[END_REF]).

Yet, this study was carried out during baseflow period (July to October) when the quality and quantity of the DOC in waters are most stable [START_REF] Chupakov | High resolution multi-annual riverine fluxes of organic carbon, nutrient and trace element from the largest European Arctic river, Severnaya Dvina[END_REF][START_REF] Holmes | Seasonal and Annual Fluxes of Nutrients and Organic Matter from Large Rivers to the Arctic Ocean and Surrounding Seas[END_REF][START_REF] Manasypov | Seasonal dynamics of organic carbon and metals in thermokarst lakes from the discontinuous permafrost zone of western Siberia[END_REF][START_REF] Neff | Seasonal changes in the age and structure of dissolved organic carbon in Siberian rivers and streams[END_REF][START_REF] Olefeldt | Effects of permafrost and hydrology on the composition and transport of dissolved organic carbon in a subarctic peatland complex[END_REF][START_REF] Tang | Drivers of dissolved organic carbon export in a subarctic catchment: Importance of microbial decomposition, sorption-desorption, peatland and lateral flow[END_REF]. Our working hypothesis is that the BDOC decreases along the HC (with the preferential degradation of easily biodegradable C molecules) and that the amplitude of this decrease depends on landscape context and the DOM origin. To test this hypothesis, we carried out aerobic biodegradation experiments (following [START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF]) and monitored resulting changes in DOM quantity and quality. We further compared the possible contribution of DOM biodegradation along the HC to published CO2 emission from the surface waters representative of the study regions.

Materials and methods

Geographical, climatic and hydrological setting of sampling sites

In high latitude continental planes, the permafrost thaw leads to the formation of "thermokarst" (thaw) lakes resulting from the soil ice melt and the permafrost subsidence in frozen wetlands. The formation and development of thermokarst lakes appeared to follow a cycle [START_REF] Kirpotin | Western Siberia wetlands as indicator and regulator of climate change on the global scale[END_REF][START_REF] Pokrovsky | Effect of permafrost thawing on organic carbon and trace element colloidal speciation in the thermokarst lakes of western Siberia[END_REF] and is an important part of the hydrological continuum of the region (Figure 1). The cycle starts with the subsidence of thawing soil forming a depression and filled by high molecular weight OM rich thaw water. The size of the depression grows due to the ongoing subsidence of peat soil and the active abrasion of edges until it reaches a lake size (thermokarst lake) with stable edges, that can be eventually drained into another lake or streams and rivers. The vegetation can then colonize the drained lake bottom and gradually the permafrost will be renewed, and the cycle can start over again by the formation of a wet depression. Lichens mostly dominate in mounds, while sphagnum type colonizes depressions, and graminoïds can be found in fens and drained lakes.

The two selected study sites are located in the artic/subarctic region, within a discontinuous permafrost area. Their detailed description can be found elsewhere [START_REF] Åkerman | Thawing permafrost and thicker active layers in subarctic Sweden[END_REF][START_REF] Brown | Circum-Arctic map of permafrost and ground-ice conditions[END_REF][START_REF] Raudina | Permafrost thaw and climate warming may decrease the CO2, carbon, and metal concentration in peat soil waters of the Western Siberia Lowland[END_REF]. The Siberian study site (referred to as Khanymey, Figure S1 A in supplementary), is located near the town of Khanymey (63°47' N;

75°32' E) in a northern taiga, with a mean annual temperature of -5.6 °C and precipitation of 540 mm. The palsa peat bogs have a peat thickness of 0.1 to 1.4 m and the active (unfrozen) layer thickness (ALT) is around 90 and 215 cm under the surface of mounds and hollows, respectively. The vegetation on the mounds is dominated by dwarf shrubs (Ledum ssp., Betula nana, Andromeda polifolia, Vaccinium ssp., Empetrum nigrum), lichens (Cladonia ssp., Cetraria, Ochrolechia) and mosses, while the hollows are covered by moss-sedge associates (grasses Eriophorum russeolum, E. vaginatum, Carex rotundata, C. limosa, Menyanthes trifoliate, Comarum palustre; mosses Sphagnum. balticum, S. majus, S. lindbergii, S. warnstorfii) and dwarf shrubs, such as Oxycoccus palustris [START_REF] Raudina | Permafrost thaw and climate warming may decrease the CO2, carbon, and metal concentration in peat soil waters of the Western Siberia Lowland[END_REF]. In Khanymey, we have chosen seven locations that belonged to a HC. They were sampled in July 2018 and used as substrates for incubation. We sampled two supra-permafrost waters from peatland, one on a mound and one on a hollow (see sampling details in [START_REF] Raudina | Permafrost thaw and climate warming may decrease the CO2, carbon, and metal concentration in peat soil waters of the Western Siberia Lowland[END_REF]; two lakes: organic-rich Chernoe and organic-poor Trisino; two streams: the one close to the outlet of the lake and another one located far from this outlet (Lymbydyakha); and finally one medium-size river Pyakopur that flows through peatland and forest [START_REF] Ala-Aho | Permafrost and lakes control river isotope composition across a boreal Arctic transect in the Western Siberian lowlands[END_REF].

Localization, surface, depth and physicochemical parameters of the seven sampled locations are presented in the supplementary Table S1.

The second site is the Stordalen mire (68°21' N and 19°02' E) located in the vicinity of Abisko in Northern Sweden (Figure S1 B). The ALT in the mire is approximately 0.5 to 1 m thick [START_REF] Åkerman | Thawing permafrost and thicker active layers in subarctic Sweden[END_REF][START_REF] Klaminder | An explorative study of mercury export from a thawing palsa mire[END_REF] and composed of 0.5 m of peat [START_REF] Malmer | Vegetation, climatic changes and net carbon sequestration in a North-Scandinavian subarctic mire over 30 years[END_REF] that overlays silty lacustrine sediment that has a glacial origin at depth [START_REF] Klaminder | An explorative study of mercury export from a thawing palsa mire[END_REF]. The mean annual temperature is -0.7 °C [START_REF] Kohler | A long-term Arctic snow depth record from Abisko, northern Sweden, 1913-2004[END_REF] and the annual precipitation is 299 mm [START_REF] Petrescu | Modelling CH4 emissions from arctic wetlands: effects of hydrological parameterization[END_REF]. The Stordalen mire is an ombrotrophic palsa that rises above semi-wet and wet minerotrophic fens, streams and shallow lakes [START_REF] Wik | Multiyear measurements of ebullitive methane flux from three subarctic lakes[END_REF]. The hummock (i.e. the equivalent of mound structure in the Khanymey site) vegetation is dominated by lichens (Cladonia spp. and Cetraria spp. and mosses, such as Sphagnum balticum (Russ.) C. Jens.) in the bottom layer while Eriophorum vaginatum and Empetrum hermaphroditum dominated in the field layer. In the hollows, the field layer is dominated by graminoids such as Carex rotundata, E. vaginatum and E. angustifolium while the bottom layer consists of mosses, such as S. balticum, S. fuscum and carpet Bryatae [START_REF] Malmer | Vegetation, climatic changes and net carbon sequestration in a North-Scandinavian subarctic mire over 30 years[END_REF]. Seven locations were sampled in the Stordalen mire in October 2018, before snowfall and ice-on: the soil solution of a hummock, the water from a crack at the edge of a small pond, the small pond itself, a fen, a lake (Villasjön, [START_REF] Wik | Multiyear measurements of ebullitive methane flux from three subarctic lakes[END_REF]) and two streams, a proximal one, near the outlet of the lake and a distal one located at the border of the mire.

Localization, surface, depth and physicochemical parameters of the seven sampled locations are presented in Table S1. In the case of soil solution samples, a pit was made on hummock (/mound) or hollow. After a few minutes, gravitational (supra-permafrost) water from the surrounding soils was accumulated in the pit. This supra-permafrost water has been used for the experiment and named "soil solution" in the rest of this study.

Experimental setup

Each water sample was collected directly in a MilliQ-rinsed Nalgene ® bottle and processed in less than 12 h after sampling following the protocol of [START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF] and [START_REF] Shirokova | Humic surface waters of frozen peat bogs (permafrost zone) are highly resistant to bio-and photodegradation[END_REF], employing the same filter units and membranes. All the manipulations were performed under sterile conditions. Waters collected in the field were first filtered through pre-combusted (4 hours at 450°C) GF/F filters (Whatman, nominal pore diameters: 0.7 µm, diameter 47 mm) using sterile filtration units (Millipore, 250 mL) and then incubated in sterile 60 mL amber glass bottles at 20 ± 2 °C, without light exposure. To keep oxygenic conditions in incubation bottles, bottle caps were not completely tightened and bottles were manually shaken at least every two days. Each sampling included filtration through pre-combusted GF/F filters mounted on a Sartorius filter unit (25 mm diameter). The duration of incubation depended on field logistics and ranged from 30 days in Siberian waters to 15 days in Northern Sweden.

Information on sampling date, replicate and the type of conducted analyses are listed in Table S2.

Analyses

The pH (uncertainty of ± 0.01 pH units), conductivity (± 0.1 µS cm -1 ) and absorbance (250 to 500 nm, 1 nm step; SpectraMax M5e Molecular Devices in Stordalen, and Eppendorf BioSpectrometer® basic in Khanymey) were measured within 1 h after the sampling. Fixation and/or storage methods for each type of delayed analysis are listed in Table S2. Water samples for DOC and fluorescence analyses were adjusted around pH = 2 using HCl, which removed the dissolved inorganic carbon (DIC) and minimize the quenching of fluorescence caused by metal complexation [START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF]. Fluorescence was measured according to [START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF] on a SpectraMax M5e. DOC and DIC were measured by a high-temperature thermic oxidation method using a Shimadzu TOC-VSCN analyzer, with an uncertainty of 2%.

Organic acids and anions were measured by high-performance ionic chromatography (Dionex Ics-5000 + ). Total bacterial cells (TBC) concentration was measured by flow cytometry (Guava® EasyCyte TM systems, Merck) using 1 µL of 10 times diluted SYBR GREEN (Merck) marker, added to 250 µL of each sample before analysis. The typical uncertainty on these measurements was between 10 and 20%.

Data treatment

The biodegradability of DOC (BDOC) was determined using commonly used equations proposed by [START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF] (Table S3). This gives the relative proportion of the biodegradable DOC in relation to the initial DOC concentration (Table S3). The apparent removal rate of DOC (RDOC) was calculated as the slope of linear regression for the incubation time (RDOC, Table S3). As the biodegradation of Khanymey and Stordalen did not last the same amount of time (respectively 30 and 15 days), in the rest of the article the RDOC and BDOC values were calculated at 15 days of incubations to allow a direct comparison between the two sites (i.e. BDOC15 and RDOC15). Absorbance data [START_REF] Peacock | UV-visible absorbance spectroscopy as a proxy for peatland dissolved organic carbon (DOC) quantity and quality: considerations on wavelength and absorbance degradation[END_REF][START_REF] Weishaar | Evaluation of Specific Ultraviolet Absorbance as an Indicator of the Chemical Composition and Reactivity of Dissolved Organic Carbon[END_REF] were used to assess the (i) the molecular weight of the DOC by the weight average molecular weight index (WAMW), (ii) the humification of DOM by the E2:E4 ratio and (iii) the relative amount of aromatic components by the specific UV absorbance at 254 nm (SUVA254) (Table S3). The Fluorescence index (FI) [START_REF] Mcknight | Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity[END_REF], was calculated to separate DOC from a terrestrial origin (FI<1.4) than DOC from a microbial origin (FI > 1.4, [START_REF] Cory | Effect of instrument-specific response on the analysis of fulvic acid fluorescence spectra[END_REF][START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF] as detailed in Table S3.

Statistical treatment of the data included least-square linear regression and Pearson correlation to assess the evolution of parameters during incubation. To compare the biodegradation in waters within and between each HC principal component analysis (PCA) was used. Input data included initial chemical and biological parameters of waters that are hypothesized to contribute to the variation of BDOC15 and RDOC15 of waters: DOC, TBC, WAMW, E2:E4 ratio, pH, PO4-P, SUVA254, conductivity and some organic acids concentrations (acetate, lactate and formate). The statistics were performed using R software (version 3.6.1) with "FactoMineR" and "factoextra" packages. All measured incubation parameters are provided in the Mendeley Data Repository (Payandi-Rolland, 2020).

Results

Inorganic water chemistry at the beginning of incubation

In Khanymey water at day 0 of incubation, the pH increased along the HC (from soil solution -mound at 4.2 to the Pyakopur river at 6.0). Specific conductivity ranged from 15 to 47 µS cm -1 in the proximal stream and in the soil solution sampled at the mound, respectively.

The DIC concentration of all waters was lower than 1.1 mg L -1 (supplementary Table S1). In Stordalen, the pH of waters increased along the HC (from 4.1 at the crack to 7.2 at the distal stream and Villasjön Lake). The specific conductivity ranged from 26 to 100 µS cm -1 in the fen and in the soil solution from the hummock, respectively. The DIC concentrations ranged from 1.7 (small pond) to 4.6 mg L -1 (proximal stream; supplementary Table S1).

Biodegradation of DOC

The DOC concentration of the Khanymey site showed high initial DOC values for waters located at the beginning of the HC (i.e. soil solution at the mound and hollow, and the Lake Chernoe, Figure 2 A.1). The DOC concentrations at the end of HC (i.e. lake Trisino, proximal and distal stream, and the Pyakopur River) were much lower and ranged between 14.5 and 16.7 mg L -1 . The DOC concentrations showed a significant linear decrease (p < 0.05, R 2 ranging between 0.45 to 0.69) in all incubations, except for the soil solution from the hollow and both thermokarst lakes (Figure 2 A.1). The corresponding BDOC value increased in all incubations from day 0 to 30 (Figure 2 B.1). After 15 days of the experiment, the lowest BDOC15 value was -0.4 ± 2.3% for the soil solution from the mound and the highest was 21.9 ± 4.9% for the Trisino Lake (Figure 3). The rates of DOC removal over 15 days (RDOC15) with the highest rates in the soil solution from the hollow (0.61 ± 0.1 mg L -1 d -1 ), and the lowest in lake Chernoe (-0.11 ± 0.1 mg L -1 d -1 ) (Figure 3 and Table S5). Soil solution from the mound exhibited the highest concentration of low molecular weight organic acids (LMWOA), i.e. formate, lactate and acetate (Table S4). Initial values of bacterial cell numbers (TBC) ranged from 2.89×10 5 (Trisino Lake) to 1.43×10 6 cells mL -1 (distal stream) (Table S1). During the incubation, the bacterial concentration increased by 30% of the initial amount in the soil solution from the mound to 72% in the incubation of the distal stream (data not shown).

The DOC concentrations in the Stordalen waters were high at the beginning of the continuum (soil solution, hummock and crack), then decreased in the small pond, and became lower at the end of the continuum (fen, Villasjön Lake, proximal and distal stream) (Figure 2 A.2). The DOC concentrations significantly decreased during incubations. The corresponding BDOC values increased in all incubations from day 0 to 15 (Figure 2 B.2). The BDOC15 ranged between 10.5 -19.9% with the lowest values in the proximal stream and the crack, and the highest in the distal stream (Figure 3). The waters located at the beginning of the continuum exhibited the highest RDOC15 while the lower RDOC15 values were observed for end-members of the HC (Figure 3 and Table S5). As for the Khanymey continuum, soil solution from the hummock contained much higher initial concentrations of acetate and formate compared with other incubated waters (Table S4). During incubation, only the soil solution from the hummock showed a decrease (78%) in acetate concentrations. The initial TBC ranged from 2.26×10 3 (distal stream) to 3.76×10 4 cells mL -1 (crack) (Table S1). During the incubation period, the lowest increase in TBC was obtained for the Villasjön Lake (49%) and the highest increase was measured for the distal stream (by a factor of 25, data not shown).

DOM optical properties

In the Khanymey continuum, the SUVA254 ranged between 3.4 L mg C -1 m -1 for the proximal stream to 4.2 L mg C -1 m -1 for the river (Figure 2 C.1). The SUVA254 increased in all samples during the incubations, with the lowest increase for the soil solutionmound (14%) and highest for the proximal stream (60%). The ratio E2:E4 increased with incubation time at the beginning of the continuum (both soil solutions and the Chernoe Lake) and decreased with time in waters located at the end of HC (from the Trisino lake to the Pyakopur River) (Figure S2 A.1). Normalized molecular weight (WAMW) allowed distinguishing 2 groups of samples (Figure S2 B.1). The first one included soil solutions from the mound and hollow and the Chernoe Lake, with stable values ranging between 516 -526 and 526 -540 Da from the beginning to the end of the incubation, respectively. The second group comprised all other water types of the continuum, with WAMW values increasing from 558 -586 Da to 590 -679 Da during the incubations.

In the Stordalen hydrological continuum, the SUVA254 ranged between 2.9 and 4.9 L mg C -1 m -1 at the beginning to 3.3 and 5.1 L mg C -1 m -1 at the end of the experiment (Figure 2 C.2). An increase in SUVA254 of 12, 19 and 22% was observed during the incubation of the soil solutionhummock, proximal and distal stream. For the remaining samples of the Stordalen continuum, the increase in SUVA254 was below 10%. The evolution of the ratio E2:E4 during incubations separated the systems in two groups (Figure S2 A.2), i.e. one with stable values at the beginning of the HC (i.e. crack, small pond, fen, and soil solution), and one with oscillating values at the end of the HC (i.e. both streams and the lake). The lowest WAMW values were recorded for soil solution -hummock and crack with the initial and final values of 506-511 and 513-515 Da, respectively (Figure S2 B.2). The highest values of WAMW were obtained for the small pond and the proximal stream (initial: 625 to 716 Da, final: 662 to 779 Da). The fluorescence index (FI) of waters in the Stordalen HC ranged between 1.52 and 2.23 at the beginning of incubation for the crack and the distal stream, respectively (Figure 2 D). FI values decreased for all waters during incubation, with values < 1.40 for the crack, the small pond and the fen and slightly higher values for the proximal stream (1.67 ± 0.2) after 5 days of incubation.

PCA results

The principal component analysis (PCA) performed on data from both hydrological continuums (Figure 4 and Table S6) showed that the component number 1 (PC1) explained 34.0% of the variation, with an important contribution of conductivity, DOC concentration, RDOC15 and organic acids concentrations (acetate and formate). The PC2 explained 26.9% of the variations, with the largest contribution coming from E2:E4 ratio, SUVA254, pH and LMWOA concentration. The BDOC15 appeared to be grouped with the pH and the E2:E4 ratio, whereas lactate, DOC, and SUVA254 were on the opposite side of the plot. Acetate and formate concentrations and specific conductivity were grouped tight. No clear visual separation by study site could be seen. However, the end-member waters of both continuums were clustered in the upper left quadrant of the plot, while other waters, except the soil solution from the mound of Khanymey, were mostly located in the lower right quadrant.

Discussion

Evolution of the DOC removal rate and biodegradability along the hydrological continuum

The decreasing trend of RDOC15 values observed along the HC, and the inverse trend for BDOC15 values (Figure 3) suggest that the soil waters located at the beginning of the continuum contain rapidly processed DOC, while the lake and river waters, at the end of HC, contain slowly processed DOC. Drastic differences in RDOC15 between the beginning and the end of the HC suggest that these changes across pore waters, supra-permafrost waters, surface small ponds and depressions of frozen peatlands, are primarily controlled by water residence time [START_REF] Catalán | Organic carbon decomposition rates controlled by water retention time across inland waters[END_REF][START_REF] Obernosterer | Competition between biological and photochemical processes in the mineralization of dissolved organic carbon[END_REF]. Indeed, when the residence time of water is short, the decay rate of OC is believed to be governed by photo-degradation and flocculation of OC [START_REF] Catalán | Organic carbon decomposition rates controlled by water retention time across inland waters[END_REF][START_REF] Evans | Variability in organic carbon reactivity across lake residence time and trophic gradients[END_REF]. Moreover, the labile parts of the DOM are likely to be firstly processed during the transition because the fresher DOC is delivered into the early stages of the HC.

The BDOC15 of streams and rivers in both continuums ranged between 10 to 20% (Figure 3) which is generally consistent with the values obtained for Arctic riverine systems (typically 10 to 40%, [START_REF] Holmes | Lability of DOC transported by Alaskan rivers to the Arctic Ocean[END_REF][START_REF] Mann | Controls on the composition and lability of dissolved organic matter in Siberia's Kolyma River basin[END_REF][START_REF] Wickland | Biodegradability of dissolved organic carbon in the Yukon River and its tributaries: Seasonality and importance of inorganic nitrogen: BIODEGRADABLE DOC IN THE YUKON RIVER[END_REF] and for waters of thawing and collapsing permafrost (10 to 40%, [START_REF] Abbott | Elevated dissolved organic carbon biodegradability from thawing and collapsing permafrost: Permafrost carbon biodegradability[END_REF]. Soil solutions, crack, small pond, fen and lake waters had a BDOC15 ranging between 0 and 20%, which is consistent with the biodegradability of lake waters from the Stordalen mire (10 to 20%, [START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF]. However, the biodegradability of fen leachate in the latter study ranged between 50 and 60%, which is 4 times higher than our values for the fen (~15%). Presumably, the fresh leachate used by [START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF] contained a higher concentration of bioavailable OM compared with natural, partially processed, fen waters used in this and other studies (i.e. [START_REF] Michaelson | The character and bioactivity of dissolved organic matter at thaw and in the spring runoff waters of the arctic tundra North Slope, Alaska[END_REF][START_REF] Spencer | Detecting the signature of permafrost thaw in Arctic rivers[END_REF][START_REF] Wickland | Dissolved Organic Carbon in Alaskan Boreal Forest: Sources, Chemical Characteristics, and Biodegradability[END_REF]. The BDOC15 values across the two continuums of this study are at the highest range of those reported in the HC of frozen peatlands of Northern Eurasia (between 0 to 10% for depression, thermokarst lake, river and stream, [START_REF] Shirokova | Humic surface waters of frozen peat bogs (permafrost zone) are highly resistant to bio-and photodegradation[END_REF] and also slightly higher than the BDOC of waters from discontinuous permafrost (5 to 15%, mean 14%) [START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF].

The PCA analysis allowed distinguishing two trends of biodegradability behavior linked to LMWOA: i) the BDOC15, which negatively correlated with lactate but was not linked to acetate and formate, and ii) the RDOC15; positively correlated to acetate and formate but which did not correlate with lactate. This could suggest that the initial concentration of acetate and formate influences the removal rate of DOC, because their presence triggers intensive microbial metabolism as it was also observed by [START_REF] Cappenberg | Carbon flow across the sediment-water interface in Lake Vechten, The Netherlands[END_REF]. In contrast, the overall biodegradability of waters may be linked to the pattern of lactate which dominated in concentrations of LMWOA of both continuums.

Evolution of the DOC quality along the hydrological continuum

The positive and negative correlation between E2:E4 spectral ratio and SUVA254 respectively, with BDOC15 observed by the PCA analysis (Figure 4), implies that an increase in the humification of DOM and a decrease in the concentration of aromatic components increase the biodegradable potential of DOM. The grouping of individuals waters located at the end of both continuums in the upper left quadrant of the PCA biplot suggests that they are influenced by the same variables (WAMW, TBC, pH and E2:E4 ratio). This is not observed for waters at the beginning of continuums, whose signals were widely spread on the biplot. Such a grouping of waters can be due to the mixing of large water bodies, receiving mostly lateral surface flow due to precipitation, while smaller water bodies are mainly supplied by lateral surface and supra-permafrost flow from thawing permafrost (Figure 1).

For the Stordalen continuum, a decreasing trend of FI in the course of incubation of each water of the HC (Figure 2 D) indicates either i) an increase in the terrestrially-derived OM or ii) a decrease in the microbially-derived OM [START_REF] Cory | Effect of instrument-specific response on the analysis of fulvic acid fluorescence spectra[END_REF][START_REF] Mann | Controls on the composition and lability of dissolved organic matter in Siberia's Kolyma River basin[END_REF]. As no fresh vegetation leachate was added in our incubations, the observed decrease of FI values during the incubation of Stordalen waters corresponds to a relative decrease in the amount of microbially-derived OM compared to the overall amount of OM. This is at odds with the reported resistance of the bacterially-derived OM to the microbial degradation in freshwater [START_REF] Kawasaki | Bacterial Contribution to Dissolved Organic Matter in Eutrophic Lake Kasumigaura, Japan[END_REF]. We, therefore, hypothesize that the relative decrease of the microbiallyderived OM occurred due to a lack of easily biodegradable terrestrial OM and the persistence of the most recalcitrant part of terrestrial OM, which is consistent with an increase in SUVA254 and WAMW with the incubation time. Moreover, a recent study on the evolution of the DOM along a soil-stream-river continuum [START_REF] Hutchins | The Optical, Chemical, and Molecular Dissolved Organic Matter Succession Along a Boreal Soil-Stream-River Continuum[END_REF] demonstrated that soil-stream waters were a hot spot of DOM degradation, with selective removal of low molecular weight (LMW) components, whereas stream-river waters were more dominated by the degradation of humic-like aromatic components. This trend is also observed in our study, particularly for the Khanymey continuum in which the E2:E4 ratio (a proxy for humification degree) increased during incubation of waters located at the beginning of the HC and decreased with time for the waters at the end of the HC (Figure S2 A.1). This is also in agreement with the rapid uptake of LMWOA observed during incubation (Table S4). SUVA254 of the Khanymey and Stordalen HC (up to 6.3 and 5.5 L mg C -1 m -1 , respectively) are greater than most of the previously reported data of waters from depressions, lakes and river of discontinuous permafrost area (3.3 to 4.4 L mg C -1 m -1 [START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF][START_REF] Shirokova | Humic surface waters of frozen peat bogs (permafrost zone) are highly resistant to bio-and photodegradation[END_REF], but comparable to the values of permafrost leachate (6.6 ± 0.1 L mg C -1 m -1 , [START_REF] Roehm | Bioavailability of terrestrial organic carbon to lake bacteria: The case of a degrading subarctic permafrost mire complex[END_REF]2009). Such high SUVA values reflect the dominance of allochthonous DOM of peat, providing the majority of DOC input to the water bodies.

Comparison between the hydrological continuum of Khanymey and Stordalen

We note that there are somewhat higher levels of BDOC15 in waters from the Stordalen peat mire compared with the Khanymey peatlands (10 to 20% and 0 to 20%, respectively, Figure 3). This difference cannot be attributed to the difference in seasons of sampling (July in Khanymey and October in Stordalen) because, even if seasons are known to play an import role in the BDOC, it has been shown that BDOC generally decreases as the Arctic summer progress [START_REF] Holmes | Lability of DOC transported by Alaskan rivers to the Arctic Ocean[END_REF][START_REF] Mann | Controls on the composition and lability of dissolved organic matter in Siberia's Kolyma River basin[END_REF][START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF][START_REF] Wickland | Biodegradability of dissolved organic carbon in the Yukon River and its tributaries: Seasonality and importance of inorganic nitrogen: BIODEGRADABLE DOC IN THE YUKON RIVER[END_REF]. Therefore, the effect of seasons would produce an opposite effect to that observed in this study. Moreover, the PCA revealed that the BDOC15 is not correlated to the TBC (Figure 4). Thus, given the higher values of BDOC15 and the lower values of TBC in Stordalen compared with Khanymey, we hypothesize that the biodiversity of microorganisms in the experiment, rather than the microbial number (TBC) impact the final BDOC of waters. It has been shown that the microbial community drastically and rapidly changes between summer and winter period with a syntrophic winter community having a higher potential for mobilizing and converting complex organic matter to more labile C sources [START_REF] Vigneron | Contrasting Winter Versus Summer Microbial Communities and Metabolic Functions in a Permafrost Thaw Lake[END_REF]. Consistent with this observation, our results show a smaller increase in the SUVA254 (reflecting the presence of aromatic components, [START_REF] Hood | Sources of dissolved organic matter (DOM) in a Rocky Mountain stream using chemical fractionation and stable isotopes[END_REF]; [START_REF] Neff | Seasonal changes in the age and structure of dissolved organic carbon in Siberian rivers and streams[END_REF]) during incubation of waters from Stordalen (12 to 22% increase) compared to Khanymey (14 to 58% increase) (Figure 2 C). In comparison, the biodegradation of peat water from the European boreal zone using the experimental approach similar to that of the present study, produced an increase in SUVA254 by 7.4 ± 4.2 % [START_REF] Hulatt | Bioavailability and radiocarbon age of fluvial dissolved organic matter (DOM) from a northern peatland-dominated catchment: effect of landuse change[END_REF].

How does CO2 production from experimental DOC biodegradation relate to fieldbased fluxes estimates

The findings of this study and widely reported dominance of low-biodegradable DOC (0-15% BDOC) in large rivers and streams of the discontinuous permafrost zone [START_REF] Frey | Optical properties and bioavailability of dissolved organic matter along a flow-path continuum from soil pore waters to the Kolyma River mainstem, East Siberia[END_REF][START_REF] Vonk | Biodegradability of dissolved organic carbon in permafrost soils and aquatic systems: a meta-analysis[END_REF] suggest that 1) the majority of DOC is degraded before its arrival to large aquatic reservoirs [START_REF] Striegl | A decrease in discharge-normalized DOC export by the Yukon River during summer through autumn[END_REF], and 2) the CO2 supersaturation and emission of surface waters of frozen peatlands can be largely a result of soil, soil pore water and sediment respiration rather than an aerobic bio-and photo-degradation of DOM in the water column [START_REF] Audry | Organic matter mineralization and trace element post-depositional redistribution in Western Siberia thermokarst lake sediments[END_REF][START_REF] Deshpande | Oxygen depletion in subarctic peatland thaw lakes[END_REF][START_REF] Rocher-Ros | Stream metabolism controls diel patterns and evasion of CO2 in Arctic streams[END_REF][START_REF] Shirokova | Humic surface waters of frozen peat bogs (permafrost zone) are highly resistant to bio-and photodegradation[END_REF].

In order to quantify the potential importance of biodegradation of DOC to the atmospheric CO2 fluxes from the study systems, we assumed that the entire consumed DOC was converted into CO2. This assumption allows to determine the maximal CO2 emission possible due to the biodegradation, keeping in mind that the true value is likely lower. Thus, to assess the amount of CO2, potentially emitted due to the total mineralization of OC via biodegradation (RCO2) in each water body, we multiplied RDOC15 (Table S5) by the mean depth of the water columns (Table S1). This calculation assumes 1) negligible uptake of C for bacterial biomass growth, 2) constant biodegradation intensity over the full depth of the water column, and that 3) the entire DOC pool is available for degradation. The obtained RCO2 values decreased for both HC from headwaters to streams and rivers of both Khanymey (range: -33 ± 45 to 241 ± 72 mg C-CO2 m -2 d -1 ) and the Stordalen (9 ± 3 to 359 ± 63 mg C-CO2 m -2 d -1 ) HC (Figure 5). The median (± IQR) RCO2 values of thaw ponds and lakes of the Khanymey site (169 ± 72 mg C-CO2 m -2 d -1 , data not shown) are sizably lower than the median CO2 emissions from the western Siberia lowland (WSL) thermokarst lakes measured by floating chambers (1,101 ± 4,150 mg C-CO2 m -2 d -1 , Serikova et al. (2019)). The RCO2 of the Khanymey streams and river (49 ± 68 mg C-CO2 m -2 d -1 , data not shown) are also much lower than the median values of CO2 emissions from the WSL rivers (6,000 ± 3,900 mg C-CO2 m -2 d -1 , Serikova et al. (2018)). Similarly, the median RCO2 of pond, fen and lake of the Stordalen site (38 ± 62 mg C-CO2 m -2 d -1 , data not shown) are much lower than the median CO2 emissions from small (4-150 m²) thaw ponds of this mire (3,348 ± 1,392 mg C-CO2 m -2 d -1 , [START_REF] Kuhn | Emissions from thaw ponds largely offset the carbon sink of northern permafrost wetlands[END_REF]). This comparison clearly demonstrates that the potential contribution of DOC biodegradation to the field-measured CO2 emissions from inland waters of permafrost peatlands is typically below 10 % (Figure 5).

Therefore, the DOC biodegradation rates obtained in this study for the water column appear to be insufficient to support the observed CO2 emissions from the water surface to the atmosphere.

Several factors can contribute to this disparity. First, aerobic and anaerobic respiration of stream, lake and river sediments as well as soil water input via supra-permafrost flow (i.e., [START_REF] Raudina | Permafrost thaw and climate warming may decrease the CO2, carbon, and metal concentration in peat soil waters of the Western Siberia Lowland[END_REF], that can produce a sizeable amount of CO2, thus increasing overall C emission potential of the aquatic systems [START_REF] Macintyre | Sediment respiration drives circulation and production of CO2 in ice-covered Alaskan arctic lakes[END_REF][START_REF] Valle | Extensive processing of sediment pore water dissolved organic matter during anoxic incubation as observed by high-field mass spectrometry (FTICR-MS)[END_REF]. For example, anaerobic C mineralization of thermokarst lake sediments is fairly well established in discontinuous permafrost zone of peat bogs in western Siberia [START_REF] Audry | Organic matter mineralization and trace element post-depositional redistribution in Western Siberia thermokarst lake sediments[END_REF] and Canada [START_REF] Deshpande | Oxygen depletion in subarctic peatland thaw lakes[END_REF]. Second, the photo-degradation [START_REF] Panneer Selvam | Photo-reactivity of dissolved organic carbon in the freshwater continuum[END_REF] of OM as well as the photo-stimulation of the microbial respiration, which is known to decrease along the HC [START_REF] Cory | Interactions between sunlight and microorganisms influence dissolved organic matter degradation along the aquatic continuum[END_REF], could enhance natural CO2 emission compared to laboratory incubations. Third, the POC present in the water column in the field can sizably contribute to overall CO2 emissions from surface waters: it has been shown that, during laboratory incubations of boreal and subarctic waters, the POC is 15 times more reactive towards biodegradation than DOC [START_REF] Attermeyer | Organic Carbon Processing During Transport Through Boreal Inland Waters: Particles as Important Sites[END_REF].

Our study dealt solely with dissolved components in closed systems and was conducted in the ice-free season of arctic peatlands. Although the results of both incubations are comparable between sites, which were sampled during the baseflow summer-autumn period, additional factors need to be considered in order to fully understand the biogeochemical processing of organic carbon in natural systems. These include i) seasonality [START_REF] Gao | Riverine dissolved organic carbon and its optical properties in a permafrost region of the Upper Heihe River basin in the Northern Tibetan Plateau[END_REF]Kaiser et al., 2017;[START_REF] Li | Distribution, seasonality, and fluxes of dissolved organic matter in the Pearl River (Zhujiang) estuary, China[END_REF], notably spring snowmelt period [START_REF] Tang | Drivers of dissolved organic carbon export in a subarctic catchment: Importance of microbial decomposition, sorption-desorption, peatland and lateral flow[END_REF] and potential anoxic conditions in summer and winter [START_REF] Deshpande | Oxygen depletion in subarctic peatland thaw lakes[END_REF], ii) water retention time (Moore, 2009;[START_REF] Olefeldt | Effects of permafrost and hydrology on the composition and transport of dissolved organic carbon in a subarctic peatland complex[END_REF][START_REF] Tang | Drivers of dissolved organic carbon export in a subarctic catchment: Importance of microbial decomposition, sorption-desorption, peatland and lateral flow[END_REF], iii) vegetation cover which affects quality and quantity of OM input (Gałka et al., 2017;Kaiser et al., 2017); iv) the ratio of water volume to soil/sediment surface area which determines the rate of fresh OM input (Polishchuk et al., 2017), v) the biodegradability of the particulate organic carbon [START_REF] Attermeyer | Organic Carbon Processing During Transport Through Boreal Inland Waters: Particles as Important Sites[END_REF], and vi) redox conditions in the water column [START_REF] Deshpande | Oxygen depletion in subarctic peatland thaw lakes[END_REF] and at the water-sediment interface [START_REF] Valle | Extensive processing of sediment pore water dissolved organic matter during anoxic incubation as observed by high-field mass spectrometry (FTICR-MS)[END_REF].

Conclusions

Biodegradation experiments demonstrated a decreasing trend of DOC removal rate along the HC from soil solutions to lakes, streams, and rivers of the discontinuous permafrost zone of permafrost peatlands of W. Siberia and N. Sweden. While we hypothesized a decrease of the DOC biodegradability along the HC, the BDOC increased from the beginning to the end of the continuum. This could be linked to preferential removal of labile, low molecular weight compartments of the DOM which are likely to be firstly processed during the transition because the fresher DOC is delivered into the early stages of the HC. Low DOM biodegradability reported in this study (0 and 20%) suggests that the majority of BDOC is degraded before its arrival to a larger aquatic reservoir or even before reaching surface waters. We also suggest that the biodegradation can support part of the CO2 emissions from surface waters of these peatlands but that other sources are likely of larger importance, including soil and sediment respiration of dissolved and particulate OC, photo-degradation and photo-stimulation of the microbial respiration. Concurrent measurements of BDOC and direct CO2 emissions in the field are needed to constrain the quantitative importance of in-situ DOM degradation for CO2 fluxes to the atmosphere. Considering the fast processing of OM at the beginning of the continuum and the increasing biodegradability of waters along the HC, this study highlights the importance of accounting for the large spatial heterogeneity of aquatic environments which is needed for mechanistically modeling of C cycling in permafrost peatlands.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: DOC concentration (mg L -1 , A), BDOC (%, B), SUVA (L mg C -1 m -1 , C) of the Khanymey (.1) and Stordalen (.2) hydrological continuum; and FI evolution of the Stordalen continuum during incubation time (D). Note that the total incubation time is 30 days for Khanymey continuum and 15 days for Stordalen continuum.

Figure 3 :

 3 Figure 3: The removal rate of dissolved organic carbon (RDOC15) of waters along the hydrological continuum (HC) after 15 days of incubations (bars and left y-axes). The filled circles show the biodegradable DOC (BDOC15) after 15 days of incubations from the same HC. The waters are from 15 locations across two HC´s: one in northern Sweden (Stordalen; light grey fill for circle and bars) and one in western Siberia (Khanymey; dark gray for circle fill and bars). The sites are represented starting from the left with the upper end of the HC and the receiving systems to the right (see figure 1). The error bars denote one standard deviation.

Figure 4 :

 4 Figure 4: Biplot of the individual PCA on waters from Stordalen (circle) and Khanymey (square) hydrological continuum and variable correlation plot (black arrow).

Figure 5 :

 5 Figure 5: Left axis: Calculated potential CO2 fluxes (mean ± SD) emitted by biodegradation (RCO2) of waters from the Khanymey (black circles) and Stordalen (grey squares) hydrological continuum. Right axis: CO2 emissions (mg C m -2 d -1 , log scale) field-based estimated from other studies in Khanymey region (dark grey boxplot) for rivers (Serikova et al., 2018) and thermokarst lakes (Serikova et al., 2019) ; and in thaw ponds from the Stordalen region (light grey boxplot, Kuhn et al. (2018)). The box encloses 50% of the data and the horizontal bar marks the median. Whiskers extend to the outermost data-points. * No outermost data-points are given for those studies. ** Negative values for the minimum outermost data-point

Acknowledgments

The fieldwork in Siberia was supported by the international program INTERACT for the 2018 campaign ("BIOCARSIB"), the Russian Fund for Basic Research grant no. 19-29-05209-mk, and Russian Scientific Fund project No 18-77-10045 (fieldwork in Khanymey). The fieldwork in Sweden was supported by the international project for mobility funded by the University Paul Sabatier (Toulouse 3, France) ("BioCarZA") and by the Swedish Research Council (grant no. 2016-05275). D.P-R was supported by a Ph. D. fellowship from the French « Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation ». M.T was supported by the « Axe transverse -AST GES » of the Observatoire Midi-Pyrénées. We thank our colleagues from the Tomsk State University (TSU) for the logistic and help during the Siberian field trip.

We thank the Climate Impacts Research Centre (CIRC), Umeå University, for hosting the Swedish experimental part of this research. We are grateful to Carole Causserand (GET) for help with DIC and DOC concentrations analyses, Frédéric Julien (EcoLab) for Dionex HPLC measurements of anions and organic acids, and Joey Allen (Ecolab) as well as Joséphine Leflaive (Ecolab) for the help on flow cytometry.

Conflicts of interests

The authors declare no conflict of interest.