Patricia Henri

Dominique Rumeau
email: dominique.rumeau@cea.fr

Ectopic expression of human apolipoprotein D in Arabidopsis plants lacking chloroplastic lipocalin partially rescues sensitivity to drought and oxidative stress

Keywords: Lipocalin, Chloroplast, Apolipoprotein D, Oxidative stress, Arabidopsis ABA, Abscisic acid, APOD, Apolipoprotein D, CaMV, Cauliflower Mosaic Virus, CNX1, Calnexin 1, EGFP, Enhanced Green Fluorescent Protein, ER, Endoplasmic Reticulum, Fv, Variable fluorescence, Fm, Maximal Fluorescence, GNT1, N-acetylglucosaminyltransferase 1, KO, Knock-Out, LCNP, Plastid Lipocalin, LHCII, Light Harvesting Complex II, MetSO, Methionine Sulfoxide, NPQ, non-photochemical quenching, Overexpression, PAGE, Polyacrylamide gel electrophoresis, PCR, polymerase chain reaction, PQ, Paraquat reactive substances, TIL, Temperature-Induced Lipocalin, VDE, Violaxanthin-de-Epoxidase

Highlights

-Human APOD was introduced into Arabidopsis plants lacking chloroplastic lipocalin.

-APOD did not substitute to plastid lipocalin in low temperature and high light-induced generation of qH, the slowly reversible component of NPQ.

-APOD partially rescued sensitivity to oxidative stress.

-APOD partially rescued sensitivity to water stress.

Introduction

Lipocalins constitute an evolutionary conserved family of small proteins widely distributed in nature whose common feature is their ability to bind small hydrophobic molecules [START_REF] Flower | The lipocalin protein family: structure and function[END_REF]. Although their primary amino acid sequences are not highly conserved, they all display a repeated structurally conserved region and an eight-stranded antiparallel β-sheet that together form a compact barrel structure with a calyx-shaped ligand pocket [START_REF] Akestrom | Lipocalins: unity in diversity[END_REF]. Shape, size and properties of this pocket vary among family members in line with the different ligands that can be accommodated. A large variety of ligands including lipids, steroids as well as secondary metabolites such as vitamins, cofactors and odorants have been reported [START_REF] Flower | The lipocalin protein family: structure and function[END_REF][START_REF] Schiefner | The menagerie of human lipocalins: a natural protein scaffold for molecular recognition of physiological compounds[END_REF]. Related to their ligands, lipocalins can fulfill a wide variety of functions such as transport of small molecules, regulation of developmental processes, signal transduction and response to stress. Because of their possible involvement in various diseases including lipid disorders, neurodegenerative diseases and cancer [START_REF] Bratt | Lipocalins and cancer[END_REF][START_REF] Xu | Lipocalins as biochemical markers of disease[END_REF], human and animal lipocalins are extensively studied.

Genomic data mining demonstrated that plants also possess lipocalins, which were classified as temperatureinduced lipocalin (TIL) and chloroplastic lipocalin (LCNP1 ) [START_REF] Charron | Identification, expression, and evolutionary analyses of plant lipocalins[END_REF]. Investigation of gene regulation, proteomic analyses and reverse genetic studies have related TIL and LCNP with stress response and tolerance. TIL appeared essential for thermotolerance [START_REF] Chi | Temperature-induced lipocalin is required for basal and acquired thermotolerance in Arabidopsis[END_REF], its accumulation during cold acclimation was described in Arabidopsis leaves [START_REF] Kawamura | Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation[END_REF] and in Siberian spruce needles [START_REF] Kjellsen | Proteomics of extreme freezing tolerance in Siberian spruce [Picea obovata][END_REF]. TIL gene expression has been demonstrated to increase with heat and cold treatment in wheat and Arabidopsis [START_REF] Charron | Molecular and structural analysis of a novel temperature stress-induced lipocalin from wheat and Arabidopsis[END_REF] and peach fruits [START_REF] Zhang | Crucial roles of membrane stability and its related proteins in the tolerance of peach fruit to chilling injury[END_REF]. In Oryza sativa, a TIL encoding gene was reported to be highly up regulated under heat stress in panicles of tolerant rice cultivar [START_REF] Prasanth | Trait and marker associations in Oryza nivara and O. rufipogon derived rice lines under two different heat stress conditions[END_REF]. TIL expression was also significantly induced by a cold treatment in Medicago sativa subsp. falcata, a forage legume cold tolerant [START_REF] He | A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress[END_REF] and in a grapefruit following a conditioning treatment that enhances chilling tolerance [START_REF] Maul | Temperature conditioning alters transcript abundance of genes related to chilling stress in "Marsh" grapefruit flavedo[END_REF]. In grape berries of Vitis labruscana, salicylic acid treatment up-regulated TIL preventing postharvest loss during cold storage [START_REF] Cai | Biochemical and proteomic analysis of grape berries [Vitis labruscana] during cold storage upon postharvest salicylic acid treatment[END_REF]. In mango fruits a TIL homolog and its corresponding gene appeared to be up-regulated during a brassinolide-mediated response to cold stress [START_REF] Li | Brassinolide enhances cold stress tolerance of fruit by regulating plasma membrane proteins and lipids[END_REF]. Moreover, TIL plays a role in salt tolerance in Populus euphratica, a salt-tolerant poplar species [START_REF] Brinker | Linking the salt transcriptome with physiological responses of a salt-resistant Populus species as a strategy to identify genes important for stress acclimation[END_REF][START_REF] Abo-Ogiala | Temperatureinduced lipocalin (TIL) is translocated under salt stress and protects chloroplasts from ion toxicity[END_REF]. In tomato two TIL encoding genes have been characterized, they display different expression profiles during development and might have a role in oxidative stress response [START_REF] Wahyudi | Functional analyses of lipocalin proteins in tomato[END_REF].

Considering LCNP, it has been demonstrated that high light, dehydration, oxidative stress and abscisic acid (ABA) induced transcript and protein accumulation in Arabidopsis [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]. In wheat, cold acclimation induced LCNP mainly in freezing-tolerant cultivar while heat exposure down-regulated gene expression [START_REF] Charron | Identification, expression, and evolutionary analyses of plant lipocalins[END_REF]. In Festuca arundinacea, salt stress induced a LCNP protein level increase, which was higher in a salt-tolerant genotype [START_REF] Pawlowicz | Remodeling of chloroplast proteome under salinity affects salt tolerance of Festuca arundinacea[END_REF]. Furthermore, LCNP abundance similarly decreased in response to drought treatment in two cultivars of F arundinacea, however, after re-watering lipocalin level returned to initial level only in the high-drought-tolerant genotype [START_REF] Kosmala | Changes in the chloroplast proteome following water deficit and subsequent watering in a high-and a low-drought-tolerant genotype of Festuca arundinacea[END_REF].

In spite of the accumulating knowledge regarding the contribution of TIL and LCNP to the resistance of plants to environmental constraints little is known about their role at the cellular and molecular levels. It has been suggested that plant lipocalins prevent accumulation of hydroperoxy fatty acids and thus stop the lipid peroxidation chain reactions initiated by reactive oxygen species (ROS) generated during stresses [START_REF] Charron | The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress[END_REF][START_REF] Chi | Temperature-induced lipocalin is required for basal and acquired thermotolerance in Arabidopsis[END_REF]. In 2009, Levesque-Tremblay et al. demonstrated that when exposed to excess light, LCNP KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in LCNP overexpressing plants. [START_REF] Bocca | Arabidopsis lipocalins AtCHL and AtTIL have distinct but overlapping functions essential for lipid protection and seed longevity[END_REF] demonstrated that LCNP deficiency dramatically enhances the photosensitivity of mutants affected in lipid protection mechanisms strengthening the hypothesis that lipocalins prevent lipid peroxidation. Interestingly, recent data obtained by [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF] demonstrated a link between LCNP and qH a slowly reversible form of non-photochemical energy quenching (NPQ), a mechanism by which photosynthetic organisms harmlessly dissipate excess absorbed light energy. The authors suggested that the accumulation of peroxidized lipids observed in Arabidopsis lcnp mutants following abiotic stress is a consequence of the absence of the photoprotective NPQ mechanism enabled by the plastid lipocalin and, thus, that LCNP might function in preventing the formation of peroxidized lipids. In addition to or as part of its role in NPQ, it is suggested that LCNP might detoxify peroxidized lipids directly [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF].

Phylogenetic studies established that plant lipocalins share similarities with the bacterial lipocalin, the mammalian apoliprotein D (APOD) and the insect Lazarillo protein. As observed in plants, the correlation between lipid oxidation status and lipocalin abundance was reported for animal and human organisms. For instance, in mice, loss of APOD increased lipid peroxidation in the brain (Ganformina et al. 2008). Conversely, human APOD overexpression reduced lipid peroxidation in mice and Drosophila [START_REF] Ganfornina | Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress[END_REF][START_REF] Muffat | Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila[END_REF]. Increased APOD levels in the human brain were reported under conditions that promote lipid peroxidation such as aging or Alzheimer's disease [START_REF] Terrisse | Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients[END_REF]. At the biochemical level, APOD ligands are well-known [START_REF] Rassart | Apolipoprotein D[END_REF][START_REF] Vogt | Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not bilirubin or E-3M2H[END_REF][START_REF] Eichinger | Structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein D[END_REF]) and the molecular mechanism underlying the antioxidative properties of APOD has been established. Indeed, the ability for APOD to control lipid peroxidation by catalyzing the reduction of radical-propagating lipid hydroperoxides has been characterized [START_REF] Bhatia | Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: Implications for lipid antioxidant activity and Alzheimer's disease[END_REF]. Insofar as LCNP displays structure conservation with APOD and displays similar antioxidative function, we wanted to investigate whether the lack of the chloroplastic LCNP could be offset by human APOD whose molecular properties are already established. To do so, human APOD cDNA fused to a chloroplast transit peptide encoding sequence was introduced in an Arabidopsis LCNP KO mutant line. Transgenic plants expressing APOD were challenged with different abiotic stresses. The plants displayed partial oxidative and water-tolerant phenotype suggesting that human APOD can replace in part, LCNP.

However, when investigating APOD function in thylakoids after high light stress at low temperature, it appeared that APOD could not contribute to qH, the slowly reversible form of non-photochemical chlorophyll fluorescence quenching, as described for LCNP. Reasons for this partial complementation are discussed and possible directions for future research are also proposed.

Materials and Methods

Plant Materials, Growth Conditions and Treatments

Arabidopsis plants were grown in phytotrons with an 8/16 day/night photoperiod and a light intensity of 130 µmol m -2 s -1 . Day and night temperatures were 22 and 18 °C, respectively. The relative humidity was set at 60 % and plants were cultivated at 100% relative soil water content. For drought stress, 9 plants of each line were grown in a single pot containing 120 g of a mix of soil and sand (2/1, w/w). Three week old plants were then subjected to water deprivation by withholding watering for 10 days. For the oxidative stress treatment, about 9 mm diameter leaf disks were excised from fully expanded leaf blades and floated onto a 1 µM solution of paraquat (PQ, N,N'-dimethyl-4-4'-bipyridinium dichloride, Sigma-Aldrich) dissolved in water for 8 hours.

Nicotiana benthamiana plants were grown in soil under controlled conditions in a growth chamber (14-h photoperiod at 25°C followed by a 10-h night at 20°C). Arabidopsis and N benthamiana plants were watered with a complete nutrient solution as previously described [START_REF] Rumeau | New subunits NDH-M, -N, and -O, encoded by nuclear genes, are essential for plastid Ndh complex functioning in higher plants[END_REF].

The lcnp KO mutant line (lcnp-1) was available through the European Arabidopsis stock Center at Nottingham University. The LCNP overexpressing line was provided by Dr F Ouellet from Université du Québec (Montréal, Canada).

Chlorophyll Fluorescence Measurements

Chlorophyll fluorescence from leaf disks was measured at room temperature using a PAM-2000 modulated fluorometer (Walz, http://www.walz.com) as previously described [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF]. The maximal quantum yield of photosystem II photochemistry was measured as

Fv/Fm = [Fm -F0] / Fm
where Fm is the maximal fluorescence level (measured using a 800 msec pulse of saturating light) and F0 is the initial fluorescence level (measured after a 2 sec pulse of far-red light).

For the comparison of the different experimental groups, statistical differences of means were analyzed with the Student's t or ANOVA tests. Letters have been used to mark statistically identical groups of means. P values are given in the legend of figure for each group of means.. Chlorophyll fluorescence imaging was done with a laboratory-built instrument as described by Johnson et al. [START_REF] Johnson | A new setup for in vivo fluorescence imaging of photosynthetic activity[END_REF].

RT-PCR

Total RNA extraction, cDNA preparation, and RT-PCR were performed as previously described [START_REF] Fabre | Characterization and expression analysis of genes encoding alpha and beta carbonic anhydrases in Arabidopsis[END_REF]. Expression of APOD was analyzed by RT-PCR with specific primers TepGW and GWapoD (Additional file 1). As control, RT-PCR was performed using actin gene-specific primers (At2g37620, Additional file 1).

APOD and Actin fragments were amplified using the following parameters: initial denaturation at 94°C for 3 minutes, 27 cycles of 94°C for 30 seconds, 60°C for 20 seconds and 72°C for 30 seconds, followed by a final extension at 72°C for 2 minutes.

Construction of Transgenic Plants

DNA sequences, which encode human APOD and the putative transit peptide of LCNP from Arabidopsis (At3g47860) were amplified by polymerase chain reaction (PCR). The primers used for PCR are indicated in Fig. 1A, their sequences are listed in Additional file 1. The final PCR product displayed attB recombination sites, which allowed cloning in pDONR201 vector using a Gateway BP Clonase enzyme mix (Invitrogen). One entry clone was fully sequenced before subsequent cloning in the binary Gateway destination vectors pB2GW7 and pB7FWG2 (Plant Systems Biology, VIB-Ghent University, Belgium; [START_REF] Karimi | GATEWAY vectors for Agrobacterium-mediated plant transformation[END_REF]) by using a Gateway LR Clonase enzyme mix (Invitrogen). The pB2GW7 vector allows expression of the cloned sequence and pB7FWG2 vector allows fusion of EGFP with the APOD C-terminus. For both vectors, the cDNA is placed under the control of the 35S CaMV promoter and of the 3′ untranslated transcribed region of a nopaline synthase gene. The binary constructs were introduced into the Agrobacterium tumefaciens strain C58. The resulting bacterial culture containing the recombinant pB2GW7 was used to transform Arabidopsis ecotype Columbia-0 (Col0) the standard flower dip method [START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF]. Transgenic plants were selected in soil after Basta treatment. The recombinant Agrobacterium strain containing the pB7FWG2 construct was infiltrated in Nicotiana benthamiana leaves according to the protocol described by [START_REF] Leuzinger | Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins[END_REF].

Chloroplast Purification and Chlorophyll Measurement

Intact chloroplasts from Arabidopsis leaves were extracted and purified by isopycnic centrifugation according to Seigneurin-Berny et al. [START_REF] Seigneurin-Berny | Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation[END_REF]). All operations were carried at 5°C. In brief, Arabidopsis leaves (100 g, from 5 week-old plants) were ground in 400 mL of grinding medium (20 mM, Tricine, pH 8.4 containing 0.4 M, Sorbitol; 10 mM, Na2-EDTA; 10 mM, NaHCO3; 0.1% [w/v] Bovine serum albumin) using a Waring blender. Chloroplasts were collected by low speed centrifugation at 2070 x g for 2 min. The chloroplasts in the pellet were re-suspended in washing buffer (20 mM, Tricine, pH 7.6; 0.4 M, Sorbitol; 2.5 mM, MgCl2; 1.25 mM, Na2-EDTA) and loaded on the top of a preformed continuous Percoll gradient. Intact chloroplasts, which concentrated in a green band at the lower part of the gradient were recovered and rinsed with washing buffer. The upper part of the gradient contains broken chloroplasts. Following low speed centrifugation (2070 x g for 2 min), chloroplasts were osmotically lysed in 10 mM Tris-HCl, pH 8. The thylakoid membranes were collected by centrifugation. Stromal proteins, which remained in the supernatant were acetone precipitated.

Thylakoids were resuspended in hypotonic buffer containing 200 mM NaCl and finally concentrated by low speed centrifugation.

Protein Extraction, SDS-PAGE and Western Blotting

Preparation of a total protein extract from Arabidopsis leaves was performed according to [START_REF] Rumeau | New subunits NDH-M, -N, and -O, encoded by nuclear genes, are essential for plastid Ndh complex functioning in higher plants[END_REF].

Following grinding in liquid nitrogen, the powder was resuspended in extraction buffer (10 mM Tris-HCl pH 8 containing 20 mM NaCl, 2 mM Na2-EDTA, 5 mM Dithiothreitol). The extract was filtrated trough two layers of muslin and the proteins were acetone (80 %; v/v) precipitated. Denaturing SDS-PAGE was performed as described by [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 13 % acrylamide gels. Proteins were either stained with BioSafe Coomassie (BioRad) or electro transferred onto 0.45 µm nitrocellulose membrane (Biotrace Pall) and probed with antibodies. Antibodies against APOD were purchased at Sigma-Aldrich/Merck. Immunocomplexes were visualized with 1:5000 diluted anti-rabbit IRDye800 antibody (Invitrogen). Detection was enabled by the Odyssey infrared imaging system.

Confocal Microscopy Observation

Pieces of N. benthamiana leaves were sampled and mounted in water. Imaging was performed using a Zeiss LSM 780 confocal laser scanning microscope and either a 20x (Plan-Apo) or a 40x (C-Apo Corr FCS) objective.

For EGFP and mCherry fluorescence analysis, the 488 nm excitation line of an argon laser was used and the fluorescence signal was detected using an emission band width of 490-530 for EGFP and of 590-620 for mCherry.

Lipid peroxidation assay

The level of lipid peroxidation products was estimated using the thiobarbituric acid (TBA) test, which determines thiobarbituric acid-reactive substances (TBARS) [START_REF] Heath | Photoperoxidation in isolated chloroplasts. I. kinetics and stoichiometry of fatty acid peroxidation[END_REF]. TBARS content was spectrophotometrically determined in Arabidopsis leaf disks (200 mg) from wild-type, transgenic and mutant plants as reported by [START_REF] Sunkar | Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance[END_REF].

Results

Targeting the human APOD to Arabidopsis thaliana chloroplast: gene expression and protein localization.

To determine whether APOD is functionally orthologous to LCNP we took an in vivo complementary approach expressing the human APOD encoding sequence in Arabidopsis. In order to target APOD to the chloroplast where LCNP is located, the putative transit peptide encoding sequence of LCNP was fused to the cDNA coding for APOD. The 20-residu APOD signal peptide, which is processed in the human mature APOD form was removed in the plant construct (Fig. 1A). The chimeric construct under the control of the Cauliflower Mosaic virus (CaMV) 35S promoter was introduced in an Arabidopsis mutant line lacking LCNP [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]. Primary transgenic Arabidopsis plants were identified following Basta selection treatment of oneweek old seedlings. In the T2 generation, 5 independent lines containing a single insertion were selected by analyzing the segregation ratio of herbicide resistance. Seeds were collected from homozygous T3 plants and used for further experiments.

The 5 transformant lines a661, a351, a421, a551, and a212 showed no phenotypic changes compared to wild type. In the five transgenic lines, APOD expression was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR, Fig. 1B). Two lines a661 and a421 exhibited a higher expression as compared to the three other lines. At the protein level, immunoblot analysis was conducted to detect the presence of APOD (Fig. 1C).

A strong band was observed in the two transgenic lines containing the higher amount of APOD transcripts (a661 and a421) while the protein was hardly detectable in the three other lines. The molecular weight of the band was estimated at 23 KDa, which does not correspond to the molecular weight of APOD estimated at 19.3 KDa suggesting either that the transit peptide was not properly processed or that APOD was post-translationally modified.

Within chloroplasts, LCNP was localized in the thylakoid lumen using a cellular fractionation approach [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]. To investigate APOD subcellular localization we performed western-blot experiments on plastid sub-fractions isolated from chloroplasts purified by Percoll gradient centrifugation (Fig. 2). APOD appeared to be of low abundance in the total cell extract. Its concentration increased only slightly in the chloroplast fraction, probably because the enrichment of proteins from percoll-purified chloroplasts in this fraction compared with total protein was low. However, we observed a larger amount of APOD in both chloroplast compartments, thylakoids and stroma. In NaCl stripped thylakoids APOD could not be detected suggesting that the protein is ionically attached to the membranes. These results suggest that in chloroplasts, APOD remains soluble in the stroma and also ionically attaches to the thylakoids.

To investigate further the localization of APOD in transgenic plants, the protein was tagged with EGFP at its Cterminus and transiently expressed using the CaMV 35S promoter in N. benthamiana leaves (Fig. 3). Six days later, leaf epidermal cells were observed by confocal microscopy. EGFP fluorescence coincided strongly with chlorophyll autofluorescence (Fig. 3A and3B) indicating that, as expected, APOD:EGFP fusion product was associated with the chloroplast. In particular, confocal imaging revealed a strong fluorescence within the guard cell chloroplasts (Fig. 3B). Furthermore, EGFP fluorescence was also detected in other subcellular structures throughout the cell (Fig. 3A). To identify EGFP labeled structures co-localization experiments were performed with different compartment markers. Constructs containing mCherry fused either to calnexin1 (CNX1) or N-acetylglucosaminyltransferase1 (GNT1) were used as markers of endoplasmic reticulum network (ER) and Golgi, respectively (Batistic, 2012, Fig. 3C and 3D). Fluorescence was evident in the ER and in Golgi. E-GFP fluorescence was also detected in nuclei (Fig 3D) but only in a few cells suggesting that it is non-specific signal.

Indeed it has been reported that GFP may translocate to the nucleus due to unspecific diffusion [START_REF] Seibel | Nuclear localization of enhanced green fluorescent protein homomultimers[END_REF]. In contrast co-localization experiments performed with a marker of plasmalemma and tonoplast excluded the presence of proteins in these membranes (data not shown).

Effect of APOD expression on sustained photoprotective energy dissipation

Recently, it has been clearly demonstrated that LCNP contributed to qH, a NPQ component, which operates under stress conditions such as cold and high light and appeared to be photoprotective [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF].

Whether APOD can replace LCNP in this function was investigated. Detached leaves of transgenic plants expressing APOD were exposed to high light at low temperature for 6 hours and maximal fluorescence Fm was recorded. The suppressor of quenching 1 mutant (soq1) with enhanced qH whose phenotype is reversed when LCNP is lacking was included in the experiment. Before the stress, wild-type plants (Col0) and the different mutant and transgenic lines displayed similar maximal fluorescence parameter (Fm, data not shown). After stress (Fig. 4), as expected since already described by [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF], soq1 displayed a lower Fm compared with Col0 while Fm in lcnp was higher than in the wild-type plant leaves. The photoprotective function of LCNP was observed in the leaves of the transgenic line overexpressing LCNP with a maximal chlorophyll fluorescence parameter lower than in Col0. In comparison, a661 and a421 displayed a Fm similar to that recorded in lcnp indicating that APOD expression cannot protect the thylakoid membrane as LCNP does.

Effect of APOD expression on abiotic stress tolerance

The chloroplast lipocalin plays an important role in tolerance to environmental constraints. In Arabidopsis, it has been demonstrated that a high level of LCNP helped plants to cope with environmental abiotic stresses such as drought, high light, cold acclimation and oxidative stress. Conversely plants lacking LCNP exhibited more damage under abiotic stresses [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]. In order to investigate whether APOD could also confer stress tolerance and play a role similar to that of LCNP in protecting plants, the transgenic lines (a661 and a421) expressing APOD in the LCNP KO background were exposed to different stress conditions including oxidative stress (Fig. 5) and drought (Fig. 6).

Since previous studies indicated that LCNP is involved in the protection of the photosynthetic apparatus against ROS, we first tested resistance of plants against cellular oxidative damage by applying the herbicide paraquat (PQ, methyl viologen) to leaf disks segments from transgenic plants. The electrons produced during photosynthesis reduce PQ and free oxygen radicals are formed initiating an oxidative stress and quickly damaging the photosynthetic apparatus. Leaf disks were exposed to 1 µM PQ for 8 hours under a light source.

Functional damage was monitored by measuring the variable fluorescence (Fv)/maximal fluorescence (Fm) values representing the maximal yield of photochemistry in the photosynthetic process (Fig. 5). The control and lcnp mutant completely lost their photosynthetic function. As expected the LCNP O/E line was significantly more resistant to PQ treatment. Interestingly, plants expressing APOD (a661 and a421) exhibited a moderate resistance while a212, a line exhibiting a very low expression of APOD displayed an oxidative stress-sensitive phenotype similar to the wild type (Col0). Considering these results, we can conclude that the constitutive presence of APOD confers enhanced tolerance to PQ-induced oxidative stress. However oxidative tolerance did not appear as notable as that recorded in LCNP overexpressing plants.

A protective effect of APOD against a drought stress was also investigated. Arabidopsis lcnp, wild-type and transgenic seeds were planted in a mix of soil and sand. Three weeks later, water was withheld for 10 days. lcnp mutants showed severe withering symptoms as compared to wild-type plants, attesting of the great sensitivity of these plants to a water deficit. The plants overexpressing LCNP (LCNP O/E) and those expressing APOD were only slightly wilted (Fig. 4A). When plants were re-watered, none of the KO mutant plants survived (Fig. 4B). In contrast, Col0, LCNP O/E and the two lines containing APOD coped well with the stress conditions. These results suggested that APOD expression in the drought-sensitive lcnp genotype could restore tolerance to a mild water stress to the level recorded in wild-type plants.

Effect of APOD expression of lipid peroxidation in Arabidopsis plants submitted to an oxidative stress

Numerous animals and human studies have reported increase APOD expression in response to conditions that promote lipid peroxidation [START_REF] Terrisse | Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients[END_REF][START_REF] Ordoñez | Apolipoprotein D expression in substantia nigra of Parkinson disease[END_REF][START_REF] Hou | Lipid signalling in plant responses to abiotic stress[END_REF]). Moreover, it has been demonstrated that APOD functions to protect against lipid peroxidation and oxidative stress [START_REF] Ganfornina | Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress[END_REF][START_REF] Muffat | Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila[END_REF]. Its capacity to scavenge hydroxyl radicals (Zhang et al. 2018) and catalyze the reduction of hydroperoxides of arachidonic acid to the non-reactive alcohols, thereby preventing lipid peroxidation propagation by radical-driven chain reactions [START_REF] Bhatia | Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: Implications for lipid antioxidant activity and Alzheimer's disease[END_REF] has also been established.

In order to further study the beneficial role of APOD in Arabidopsis plants submitted to abiotic stresses and investigate a possible function in protecting against lipid peroxidation, Arabidopsis leaf disks were exposed to a PQ-induced oxidative stress as described in Fig. 5 [1µM PQ for 8 hours and exposed to light (130µmol m-2 s-1)]. The levels of lipid peroxides in plant tissues were determined as TBARS contents (Fig. 7). TBARS result from the reaction of peroxidized polyunsaturated fatty acids with thiobarbituric acid. Thus, they are an index of lipid peroxidation and have been used as a metabolic marker for oxidative stress [START_REF] Heath | Photoperoxidation in isolated chloroplasts. I. kinetics and stoichiometry of fatty acid peroxidation[END_REF]. As shown in Fig. 7, PQ treatment induces an increase in the TBARS content. Lcnp leaf disks accumulate a significantly higher amount of TBARS compared to the control (Col0) when exposed to the stress. Lower amounts of TBARS were observed in LCNP O/E and APOD (a421 transgenic line) treated disks indicating that the degree of lipid peroxidation was lower in these plants. However, APOD samples displayed higher TBARS level than LCNP O/E disks. These results suggest that in Arabidopsis, APOD could exert a reductive activity on plant lipid hydroperoxides and thus prevent lipid peroxide propagation as described in animals and humans.

However, this effect is not as effective as that of LCNP. This result is consistent with the effect of PQ on Fv/Fm described in Fig. 5. As previously proposed for LCNP [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]), the lower PSII damage observed in APOD transgenic plants following an oxidative stress is probably due to the ability of APOD to alleviate lipid peroxidation in Arabidopsis.

Discussion

In the present work, we developed an interkingdom complementation approach to investigate Arabidopsis chloroplastic lipocalin function. The rationale behind this approach was the assumption that human APOD because it shares similar antioxidative properties and a similar three-dimensional structure with LCNP can rescue the lack of chloroplast lipocalin in Arabidopsis. Numerous reports have demonstrated that interspecies functional complementation studies facilitate structure-function analysis [START_REF] Dolinski | Orthology and functional conservation in Eukaryotes[END_REF]Schultz and Thöny-Meyer, 2000). To the best of our knowledge, only one report addressed a functional complementation of plant protein with animal protein [START_REF] Brioudes | Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants[END_REF]). Here, we report that expression of human APOD in Arabidopsis, by means of transgenic lines, can partially compensate for the lack of the plant lipocalin LCNP.

The results are consistent with a conserved function of APOD and LCNP under stressful conditions. However, if the results obtained with the drought and oxidative stresses point to the protective effect of constitutive expression of APOD in plants lacking LCNP, this effect is not as effective as that conferred by LCNP overexpression. Moreover, when investigating APOD function in NPQ after high light stress at low temperature, it appeared that APOD could not contribute to qH as described for LCNP [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF]. It is intriguing to notice that in Arabidopsis, APOD can fulfill only certain functions of LCNP.

The appropriate subcellular localization and translocation of lipocalins are crucial to their functionality. LCNP located into the lumen of thylakoids [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]) has been shown to be involved in qH, which has been localized to the peripheral antenna of photosystem II, suggesting that LCNP could either directly form a NPQ site or indirectly modify the light harvesting complex II (LHCII) membrane environment [START_REF] Malnoë | The Plastid Lipocalin LCNP Is Required for Sustained Photoprotective Energy Dissipation in Arabidopsis[END_REF]). Lumen targeting is a complex process, which may involve two different pathways able to recognize a thylakoid-targeting signal peptide [START_REF] Albiniak | Targeting of luminal proteins across the thylakoid membrane[END_REF]). The N-terminal bipartite signal for chloroplast import and thylakoid transfer that drives the import of LCNP from the cytosol to the lumen has not yet been characterized. In our construct, LCNP transit peptide extension was defined based on unpublished data (Malnoë, personal communication). In Arabidopsis transgenic plants, APOD fused to the putative transit peptide of LCNP was clearly targeted to the chloroplast (Fig. 2 and Fig. 3). However, we could not find evidence that APOD penetrated into the lumen of thylakoids. Inappropriate processing could explain the APOD unexpected size described in Fig. 1 and the lack of APOD in the lumen.

APOD is a glycoprotein and we cannot exclude that glycosylation occurred in cells of transgenic plants. The localization of APOD fused to GFP into Golgi and ER supports this hypothesis (Fig. 3). Moreover, this posttranslational modification could explain the unexpected molecular weight of APOD in a plant cell extract (Fig. 2). Lastly, targeting of glycoproteins into chloroplasts is well known [START_REF] Faye | Novel pathways for glycoprotein import into chloroplasts[END_REF] but import into thylakoids lumen has never been described. The lack of lipocalin into the chloroplast subcompartment, although the protein is present in the stroma, may explain why APOD did not contribute to qH as LCNP does.

Moreover, it has been demonstrated that an unpaired cys residue in APOD is responsible for covalent attachment of the protein with Apolipoprotein A-II (APOA-II) in plasma [START_REF] Vogt | Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not bilirubin or E-3M2H[END_REF]. Although no homolog of APOA-II has been described in plants and since the cys residue was maintained in the plant chimeric construct we cannot exclude unphysiological crosslinking of APOD to membrane proteins in plant cells. This could account for the localization of EGFP-APOD in ER and Golgi (Fig. 3).

Molecular phylogenetic analysis of lipocalins not only revealed that the chloroplastic LCNP shared significant structural homology with the mammalian APOD [START_REF] Ganfornina | A Phylogenetic Analysis of the Lipocalin Protein Family[END_REF][START_REF] Charron | Identification, expression, and evolutionary analyses of plant lipocalins[END_REF] but also suggested that both proteins might correspond to the same overall function in terms of general nature of binding ligands since both proteins appeared in the same clade [START_REF] Charron | Identification, expression, and evolutionary analyses of plant lipocalins[END_REF][START_REF] Lakshmi | Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily[END_REF]. In vitro ligand binding assays have shown that APOD is able to bind a variety of lipids in its hydrophobic binding cavity [START_REF] Rassart | Apolipoprotein D[END_REF]. More recently, it has been clearly demonstrated that APOD displayed the highest binding affinity for arachidonic acid (AA) and progesterone [START_REF] Rassart | Apolipoprotein D[END_REF][START_REF] Vogt | Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not bilirubin or E-3M2H[END_REF][START_REF] Eichinger | Structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein D[END_REF].

Partial functional complementation of LCNP deficiency by APOD cannot be related to the nature of APOD ligands since neither AA nor progesterone are present in Arabidopsis. However, we cannot exclude the possible affinity of APOD to other structurally related lipids that may be involved in signaling cascades. Indeed, numerous signaling lipids whose synthesis can rapidly be activated upon abiotic stress signals have been identified in plants (for a review see [START_REF] Dassati | Apolipoprotein D takes center stage in the stress response of the aging and degenerative brain[END_REF]. In Arabidopsis, APOD could exert a control of the signaling lipid molecules that decipher the stress response cascade and thus modulate transgenic plants tolerance.

In plants, a similar function for LCNP cannot be excluded as well.

During last decade, several authors have published that APOD function extended well beyond the transport of lipophilic molecules. Therefore, an extensive work allowed to establish a correlation between lipid oxidation status and APOD abundance in human and mammals. Increased APOD expression was reported in the human brain under conditions that promote lipid peroxidation and up-regulation of antioxidant gene expression such as aging or neuropathological conditions including schizophrenia, Parkinson's and Alzheimer's diseases [START_REF] Terrisse | Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer's patients[END_REF][START_REF] Ordoñez | Apolipoprotein D expression in substantia nigra of Parkinson disease[END_REF][START_REF] Hou | Lipid signalling in plant responses to abiotic stress[END_REF]. Studies in mice showed that loss of APOD increased lipid peroxidation in the brain, that cerebral APOD expression is increased in response to PQ-induced oxidative stress and that human APOD expression reduced lipid peroxidation [START_REF] Ganfornina | Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress[END_REF][START_REF] Muffat | Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila[END_REF]). In addition, expression of human APOD reduced the accumulation of aldehydic end-products of lipid peroxidation in an aged Drosophila model [START_REF] Walker | Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan[END_REF][START_REF] Muffat | Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila[END_REF]. In plants, the protective role of LCNP in oxidative stress has been related to its ability to prevent or modulate lipid peroxidation caused by ROS action [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]). Thus, a LCNP KO Arabidopsis line, which exhibited serious damage upon photo-oxidative stress induced by drought, high light or PQ displayed a rapid accumulation of hydroperoxy fatty acids. In contrast, in LCNP overexpressing lines, which coped better with these conditions, the lipid peroxidation level remains very low [START_REF] Levesque-Tremblay | The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stress[END_REF]). Related to APOD antioxidative function, it has been demonstrated that APOD had the capacities to scavenge hydroxyl radicals (Zhang et al. 2018) and catalyze the reduction of hydroperoxides of AA to the non-reactive alcohols, thereby preventing lipid peroxidation propagation by radical-driven chain reactions [START_REF] Bhatia | Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: Implications for lipid antioxidant activity and Alzheimer's disease[END_REF]. This reaction involves a highly conserved methionine residue (Met93 in human APOD) located at the hydrophobic surface of the protein and results in oxidation of the methionine thioether group to form methionine sulfoxide (MetSO, [START_REF] Bhatia | Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: Implications for lipid antioxidant activity and Alzheimer's disease[END_REF].

Interestingly, APOD expression in Arabidopsis decreases PQ-induced lipid peroxidation (Fig. 7) suggesting that in plants APOD could scavenge hydroxyl radicals and limit lipid peroxidation. Thus, it can be speculated that APOD could exert its reductive activity on plant lipid hydroperoxides such as hydroperoxides of linolenic (18:3), linoleic (18:2) and roughanic (16:3) acids, the major polyunsaturated fatty acids in plants. A lower affinity of APOD for plant hydroperoxides or an insufficient amount of APOD in plant cells could account for the lower protective effect of the human lipocalin as compared to that provided by LCNP. By analogy, the protective function towards oxidative stresses conferred by LCNP could be due to a surface methionine residue, similar to APOD Met93 and able to reduce plant fatty acid hydroperoxides, as well.

In conclusion, our data indicate that despite structural homology and similar contribution to lipid oxidation status in cells, APOD when introduced in Arabidopsis cannot fully substitute for LCNP. We cannot exclude that APOD and LCNP share similar oxidative protecting mechanisms, however, APOD displays molecular features which hamper its protective function in plant. Thus, we propose that APOD may be glycosylated and able to bind other proteins in plant cell and thus unable to reach thylakoid lumen where LCNP plays its photoprotective role. APOD that remains in the cytosolic compartment is able to partially protect cells from reactive oxygen species produced during environmental stress. We suggest that this functionality may reside in a particular methionine residue, which has been demonstrated to play a crucial role in animal APOD protective activity. The findings of this study provide new leads for future research. In particular, we are examining LCNP methionine residues to assess their contribution in lipid hydroperoxide reduction. Finally, we also intent to change the unpaired APOD Cys to investigate whether APOD inability to protect photosystem II is due to its failure to reach the thylakoid lumen. I. Sequences of PCR Primers designed for the cloning of chimeric genes and transgenic plant characterization.

Authors' contributions

Color should be used for Figures 3, 4 and 6

Supplementary Table

Figure 1 .

 1 Figure 1. Molecular characterization of transgenic plants expressing a chimeric gene containing the LCNP putative transit peptide fused to APOD cDNA. A, The gene structure. Shaded and white box represent the sequences coding for APOD and LCNP transit peptide (TP), respectively. Small arrows represent PCR primers whose sequences are listed in Supplemental TableI. In the lower part, amino acid sequence of the chimeric protein is also indicated. In bold, LCNP transit peptide. B, RT-PCR analysis of transgenic plants. Total RNA was extracted from leaves and PCR was performed using TPepGW and GWapoD as gene-specific primers for the chimeric gene (left panel) and actin gene as an internal control (right panel). C, Western blot analysis. Total proteins were extracted from leaves and subjected to gel blot analysis using anti-APOD antibodies. Inset, Coomassie blue stained gel as a loading control.

Figure 2 .

 2 Figure 2. Subcellular localization of APOD. Intact chloroplasts were isolated from leaves of the a661 transgenic line by centrifugation through a Percoll gradient. Chloroplasts were lysed by hypo osmotic shock and further separated into thylakoid and stroma fractions for immunoblot analysis. Thylakoids were washed with a buffer containing 200mM NaCl. Proteins collected following acetone precipitation were separated on a 13% SDS-PAGel. A, Coomassie Blue stained gel; B, immunoblot detection of APOD in the chloroplast sub fractions.

Figure 3 .

 3 Figure 3. Co-localization of APOD-EGFP with compartmental markers. Nicotiana benthamiana leaves were agroinfiltrated for the transient expression of APOD:EGFP. Confocal scanning microscopy observations were performed at 6 days post infiltration. A, Cell expressing EGFP in various subcellular structures; B, Chloroplast marker chlorophyll autofluorescence; C, Endoplasmic reticulum marker m Cherry-CNX1; D, Golgi marker GNT1-mCherry; (s, stomata). N benthamiana infiltrations with the different constructs were reproduced several times and gave similar results.

Figure 4 .

 4 Figure 4. Fluorescence phenotype in APOD-containing transgenic lines induced by cold and high light treatment. Isolated leaves were exposed to cold (6°C) and high-light (1500 µmol photons m -2 sec -1 ) treatment for 3 hours. Representative image of maximum chlorophyll fluorescence emission (Fm) monitored with a fluorescence imaging system.

Figure 5 .

 5 Figure 5. Tolerance of Arabidopsis APOD-containing transgenic lines to oxidative treatment. Leaf disks were floated on a solution of paraquat (PQ, 1 µM) for 8 hours and exposed to light (130 µmol m -2 s -1 ). The photosystem II photosynthetic efficiency in the leaf disks was estimated by chlorophyll fluorescence determination of photochemical yield (Fv/Fm). The data represent the means ± SD (n>6). Student's t-and ANOVA-tests performed, enabled us to identify 4 groups of statistically different means (P ≤ 0.001). P values of "t" tests of groups b, c and d were 0.560, 0.977 and 0.559, respectively.

Figure 6 .

 6 Figure6. Tolerance of Arabidopsis APOD-containing transgenic lines to water deficit stress. Nine plants of each line were grown in a single pot containing a mix of soil and sand (2/1, w/w) under normal conditions for 3 weeks (A). Then watering was completely withdrawn for 10 days (B). Plants were then re-watered for 5 days (C). These experiments were repeated twice and gave similar results.

Figure 7 .

 7 Figure 7. APOD expression reduces paraquat-induced lipid peroxidation in transgenic plants. Leaf disks were floated on a solution of paraquat (PQ, 1 µM) for 8 hours and exposed to light (130 µmol m-2 s-1). Lipid peroxidation is expressed as TBARS contents. Values are given as means ± SD from three replicates and each letter represents a statistical different group (P<0.001).

Initially named CHL, the chloroplast lipocalin was renamed LCNP ["Lipocalin in the Plastid"] byMalnoë et al. in

This abbreviation will be used throughout the text for designating the plastid lipocalin.

Acknowledgements

We thank Pr Eric Rassart (Université du Québec, Canada) for the kind gift of APOD cDNA, Dr. Olivier Batistic (Universität Münster, Germany) for providing the mCherry.Calnexin1 (mCherry-CNX1), N-acetylglucosaminyl-transferase1.mCherry (GNT1-mCherry) and Two-pore-K + -channel1.mCherry (TPK1-mCherry) plasmids and Dr François Ouellet for the LCNP overexpressing line. We thank Nathalie Duong (Aix-Marseille University, Cadarache) for technical assistance with the Arabidopsis transformation, Michel Havaux for preliminary discussions and Jean Alric and Louis Dumas (UMR7265-CNRS-CEA-AMU, Cadarache) for help with Chlorophyll fluorescence imaging. We also thank the Phytotec platform (Cadarache) for growing plants under control and stress conditions. Support for the microscopy equipment was provided by the Région Provence Alpes Côte d'Azur, the Conseil Général des Bouches du Rhône, The French Ministry of Research, the CNRS and The Commissariat à l'Energie Atomique et aux Energies Alternatives. Finally we thank Xenie Johnson and Jean-Luc Montillet (CEA Cadarache) for critical reading of this manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Availability of data and material

Not applicable

Declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Conflicts of interest

The authors declare that they have no conflicts of interest.

Supplementary Table I. Sequences of PCR primers designed for the cloning of chimeric genes and transgenic plants characterization


Meriem Ouadah
email: mouadah@usthb.dz

Fatiha Merazka
email: fmerazka@usthb.dz

A Network Coding Approach for Reliable SDN Based UAV Networks

Keywords: UAV, SDN, Network Coding, NTN, Pollution Attack

Recently, the integration of unmanned Aerial Vehicles (UAV) in Software-Defined Networking (SDN) based environments has seen an increasing research turnout as it allows a logical centralization of the control function while enhancing Information Technology (IT) management. This paper focuses on the use of Network Coding (NC) to secure the exchanged data between the UAVs where we consider the deployment of an SDN-based UAV architecture in a logical environment. We also consider the integration of NC in a UAV-SDN-based architecture to reduce the number of packet retransmissions and improve network reliability in lossy networks. To further secure data integrity in our system, we propose a pollution attack detection method as a tool to identify and block malicious packets from accessing the network. We assume that the UAVs are powerful enough to perform all encoding and decoding operations during communication.

Introduction

Hardware-based networks rely on equipment such as switches and routers to handle data, by making traffic flow decisions individually, which affects network performance in terms of reliability. Recently, the dynamic architecture of Software-Defined Networking (SDN) solved this problem by separating the control and data planes in order to provide a centralized network that enables automated deployment of applications. By using controllers, it reinforces network agility, programmability and security allowing a rapid response in the case of an incident, making it a highly resilient network scheme [START_REF] Al-Gboury | Investigate and Compare Software-Defined Net-work Controllers for UAV Networks Management[END_REF].

In addition to SDN, the use of unmanned aerial vehicles (UAV) systems is growing rapidly as it allows static base stations to move freely in the environment even in previously inaccessible areas, resulting in a rich set of use cases ranging from civilian to military applications. This has led to the combination of both technologies to create UAV-SDN based systems. This latter provides high flexible mobile architectures, improves quality of service, latency, and signal losses. As in all communication networks, SDN systems may suffer from security threats and packet losses. Network Coding (NC), precisely Random Linear NC (RLNC) are usually integrated in SDN systems to help in mitigating this problem due to its packet loss concealment methods as well as to its weakly secure nature, in addition to other RLNC based secure schemes that aim to protect information against wiretappers.

Technically speaking, RLNC allows intermediate nodes to perform linear transformations (encoding and decoding operations) on the transmitted packets, instead of relaying information directly [START_REF] Goncalves | Random Linear Network Coding on Programmable Switches[END_REF] to ease network congestion as well as to improve reliability, security, load balancing, power consumption and time delivery in the network [START_REF] Manoranjitham | Performance Enhancement Using Network Coding in Dynamic Source Routing[END_REF]- [START_REF] Chu | Random linear network coding for peer-to-peer applications[END_REF]. These latter set of advantages may provide a possibility for RLNC integration in None Terrestrial Networks (NTN) such as SDN-UAV, especially in terms of energy preservation and robustness.

NTN have attracted recently a huge research focus; it is considered as the futuristic 6G networks that may meet the demands and the requirements of the upcoming technological development. Recent research in [START_REF] Mishra | Drone Networking in the 6G Era: A Technology Overview[END_REF] explored candidate technologies for integration with UAVs.The authors in [START_REF] Shakhatreh | Unmanned Aerial Vehicles (UAVs): A Survey on Civil Applications and Key Research Challenges[END_REF] are expecting UAVs to raid civil applications despite the cost they incur due to their compatibility with trendy research technologies such as cloud, SDN and machine learning.

Recently, the scientific community focused research on UAVs due to the need for optimized mobile systems to handle and relay the great amount of exchanged data in IoT networks. Precisely UAV-SDN-based systems attracted increased interest as it improves network optimization and quality of service.

In [START_REF] Suggala | SDN assisted UAV communication systems: Efficient Deployment Strategies[END_REF], a new algorithm for congestion control has been proposed and discussed jointly with UAV placements in SDN. Results showed decreased packet loss along with minimized power consumption after using multi-hop forwarding in a circular stadium architecture. Another simulation-based research in [START_REF] Colpaert | Drone delivery: Reliable Cellular UAV Communication Using Multi-Operator Diversity[END_REF] offers a 20% improved UAV coverage by applying multi-operator diversity which creates a cost-effective reliable mobile system.

SDN-based UAV systems require robustness in the network, scientists researched securing the network coding algorithms, and earlier works in [START_REF] Krohn | On-the-fly verification of rateless erasure codes for efficient content distribution[END_REF]- [START_REF] Gkantsidis | Cooperative Security for Network Coding File Distribution[END_REF] proposed sharp cryptographic approaches to secure network coding such as homographic hashing. Another approach in [START_REF] Cao | Secure Communication with Network Coding[END_REF] employed a cryptosystem combination with a secret key, and the results proved the inability to extract important information when intercepting the packets. Traditional cryptographic methods remain very expensive and difficult to implement. Therefore, the authors in [START_REF] Adat Vasudevan | On Security Against Pollution Attacks in Network Coding Enabled 5G Networks[END_REF] proposed novel security schemes for 5G networks in which they used SDN controllers to distribute the keys and blockchain to store and share them. These methods have been proven efficient in terms of flexibility and easy implementation. Another approach in [START_REF] Hayashi | Secure Physical Layer Network Coding versus Secure Network Coding[END_REF], investigated the advantages of the secured physical layer network coding (PLNC) over ordinary NC. The comparison between the two methods showed that PLNC exploits noise to maintain secrecy at intermediate nodes without the need for randomness as in NC. Although the above papers have explored different aspects of UAV, SDN and NC, they did not consider heterogeneous communication environments. This calls for novel communication network solutions to ensure high reliability. In this work, we consider the ability of adding a new layer of security to the NC algorithm by implementing a pollution attack detection algorithm.

This paper is organized as follows. Section 2 describes the proposed architecture for UAV communication networks. In Section 3, an experiment is conducted to investigate the advantages that NC induce to our network. Section 4 evaluates the effectiveness of the proposed pollution attack detection algorithm. Section 5 concludes this paper.

Proposed Solution

It is common knowledge that UAV networks bring a huge value in terms of mobility as they travel through different kinds of environments. In an SDN based UAV architecture, traffic control is performed in both a centralized and a decentralized way considering UAV vehicles as the main actors. The vehicles have the ability to communicate and exchange data with each other, with the base station or other devices if configured to do so where the decentralized function is being presented. On the other hand, when using the SDN architecture, the vehicles will operate in a centralized function instead of an individual one by taking orders from the controller. Based on this context, an approach in [START_REF] Li | UAV Communications for 5G and Beyond: Recent Advances and Future Trends[END_REF] proposed that UAVs can act as switches that collect and distribute information in the swarm while the base stations receive data, make control decisions and manage the vehicles. We take this to the next level, by making UAVs act both as controllers and switches at the same time. To simplify the explanation, The idea is to create a hybrid environment where centralized and decentralized functions can coexist as an enhancement to the wireless 6G network. This section is divided into two parts, in the first one, we describe the novel architecture, how we implemented it as well as the tools used for that. Next, in the second part, we explain the proposed mechanism that aim to add a second layer of security against pollution attacks.

The Architecture

As explained earlier, we propose the use of UAV vehicles as both controllers and switches simultaneously. Fig. 2 illustrates the function of each layer of this network architecture. The first group of UAVs is called group A, which is a set whose elements are controllers that issue orders to group B, control traffic flow and make resource allocation decisions. Group B, on the other hand is the switching group that travels through the environment, collect data and perform basic switching functions. The benefit of using a group of controllers or UAV controllers is to keep up with mobility needs, in traditional centralized networks or algorithms, there exists a single decision-making entity, which implies a direct connection of this latter to all connected devices. Since in UAV networks, the vehicles must travel freely we use many controllers to travel along with the vehicles; this way, communication will be more flexible as each entity communicate with the closest controller. Moreover, we precise that UAV controllers also communicate with base stations because they are considered the source and destination of all communications.

Simulation wise, we wanted to prove the efficiency that can be added to the wireless network using NC and other layers of security. For this purpose, we created a scenario of a pollution attack detection using a simplified version of the architecture that we explained earlier in this section. We note in what follows that each virtual machine is considered as an UAV that's circulating in the network.

The architecture constitutes of a server, an SDN controller machine, a virtual switch, two legitimate clients and an attacker as illustrated in Fig. 3. To implement it, we selected the following technologies: the GNS3 network simulator, VirtualBox hypervisor, open daylight SDN controller, Open v switch manager with a management interface and the KODO library for RLNC []. These technologies were tested apriori to verify their adaptability with the solution and the chosen environment. There is a minor twist in this architecture where we added a virtual switch only to highlight the centralized function brought by SDN. The switch is linked to the controller in order to direct all the traffic to it, as the controller machine is the one deciding the flow based on the global view of the network. We may consider the switch as a relay for the other vehicles but in a real-life scenario it is not required in the architecture. When a new node is added to the network, the controller communicated the necessary rules to the switch to execute proper data routing. The server, however, replaces the base station as a traffic destination where we add the secure pollution attack detection mechanism.

Pollution Attack Detection Mechanism

Despite using RLNC in our SDN based UAV architecture, the network still suffers form a very serious flaw, attackers controlling one of the intermediate nodes that have the ability to recode packets can easily inject corrupted packets into the network. Those packets, if undetected, will eventually be mixed with other valid packets leading to information loss. We refer to this attack as a pollution attack. In our architecture, we created a mechanism that establishes an IP address data base in the server to verify the legitimacy of the sender before decoding its encoded data thus detecting the intrusion. The diagram in Fig. 4 illustrates this solution. For the data base we defined two IP address ranges as the legitimate ranges, one for each client machine while the attacker belongs to a different IP address range. When receiving data, the server retrieves IP information from the metadata sent with the coded packets before decoding the sensitive information. It then performs two logical "And" operations one between the IP address and the subnet mask and the other between the network address in the data base and the subnet mask. It, then, compares the two results to make a decision. In the favorable case, there will be a match which means data is legitimate thus the server starts the decoding process and accepts all packets from that source. However, if the IP address does not belong to the predefined range, data is considered corrupted and a pollution attack is detected, the server will not decode any of the received packets, they will be discarded when a certain loss rate is exceeded.

NC Performance Evaluation

To investigate the enhancements brought by NC, we studied two cases, the case of using NC and the case of not using it.

For the first case, the aim was to test if the decoding process can be affected by a certain packet loss rate as it exacerbates network performance. In this architecture, packet loss can be viewed as a serious threat as it could block and influence the decoding process due to the properties of NC where an entity needs to receive a certain number of encoded packets in order to perform decoding and retrieve original data.

We proceeded by simply using our program where legitimate clients send encoded data to the server. The program was executed several times while varying the percentage of losses on the links.

To variate losses, we used the GNS3 "Packet Filter" tool that applies packet filters on the links without stopping the nodes in the network for operating. GNS3 offers three main filters which are the following:

-The drop in frequency: it deletes a packet every x packets.

-Packet loss: Allows you to drop a packet randomly.

-Latency: Used to delay the transmission of packets.

All packet filters can be combined, which means that we can apply a packet loss filter and a latency filter on the same link. For our test we used the packet loss filter only.

For the second case, we aim to investigate network performance when there is no NC in order to compare later with the first case and see if anything was added or the contrary. We proceeded by using the Iperf tool that stands for "Internet Performance working group " . The latter can measure and adjust the performance of the network by reporting the quantity of transferred data and the transmission rate. In simple words, the tool sends a defined number of uncoded packets, generates a random packet loss percentage and other statistics such as: the transmission and reception bit rate. Fig. 5 displays a line chart of the obtained results from both performance comparison tests.

From the graph, we observe that in the case of using NC, the reception bit rate value remains in a constant zone [6.5 * 10 -2 -7.5 * 10 -2 Mbytes/s] and does not decrease too much compared to the initial value even with a high percentage of losses (10%). On the other hand, in the absence of NC, we can see that the bit rate decreases seriously each time we increase the percentage of losses until it reaches a minimum value below 0.5 * 10 -2 Mbytes/s. The change of bit rate indicates that packet loss affects the performance of the network. If the network is stable when generating losses then the performance is deemed good. Otherwise, we say that the performance is unsatisfactory. We can conclude that packet losses do not affect the transmission reliability when we use NC which makes the network very efficient.

Note that the different packet loss generation was approximately the same in both cases otherwise, we could not have compared between the two of them.

Attack Detection Performance Evaluation

To evaluate the effectiveness of the security mechanism we simulated the pollution attack, as described in the architecture of our solution, we used two legitimate client machines and one attacker machine, we launched the attacker's machine and sent manipulated data to the server, the server extracted the IP address from the metadata, then compared it with the data base. Fig. 6 below illustrates the output in both cases where a match is found and a pollution attack is detected.

Based on the simulation above, we can say that the mechanism works every time and it is 100% effective in this case. If the attacker manipulates hundreds of packets they will never be accepted by the server. This means that we succeeded in adding another layer of security to our system by protecting the exchanged encoded data thus securing NC.

We precise that the received malicious packets from the attacker were not dropped instantly but were put in the buffer which drops them randomly until they are completely gone.

To check if the packets are truly discarded, we conducted an experiment that relies on two factors: packets loss rate and the algorithm's execution number. We executed the algorithm of the pollution attack detection times and kept records of the packet loss rate each time. Results are shown in Fig. 7 below. The Column chart shows that the decoded malicious packets are kept in the buffer until the packet loss reaches the value of 30% which is the turning point of the system, if the packet loss rate is below or equal to 30%, the system will not discard any packet. However, when it exceeds the value of 30%, malicious packets are immediately discarded. These results prove that the system acts intelligently by not discarding the packets until the loss rate increases because it considers it as an abnormal activity. The latter does not have to be true as the main goal of the test is to discard those malicious packets despite knowing that it is impossible for them to affect the network in any way as long as they remain in a coded form which prevents the malicious data from invading the system.

The attack described earlier is considered as an external network attack because the attacker machine does not belong to the system. Legitimate machines can also be a threat, and send malicious packets without being detected. We present the diagram in Fig. 8 below that describes the functioning of this system. The advantage of our security solution is to give network administrators the possibility of controlling access by predefining legitimate networks. This preset means that detection is 100% possible. Administrators can add as many networks as they want. However, it is impossible to detect the attack when it comes from a legitimate network in this scenario. Consequently, the system requires additional access control mechanisms to ensure the traceability of the attacker. This could be discussed in other scopes since we have already reached our goal which was originally exploring the possibilities to add new layers of security for a more reliable network.

Conclusion

There is no doubt that UAV networks are the future of wireless communications such as/ 6G networks. Enhancing reliability is probably the most fundamental asset to match the highly dynamic property of UAV networks. In our paper, we have focused on exploring the use of different technologies to enhance reliability in UAV networks. Having a very scalable architectural design, we were able to propose an improvement that is based on using a group of vehicles as controllers to keep up with the dynamic mobile nature of SDN-based UAV networks and benefit from the flexibility added by SDN. We proposed the use of the novel NC algorithm to ensure flexible data transmission and data integrity, this algorithm proved to be effective by significantly increasing network performance. Moreover, we proved the possibility of adding another security layer while keeping this harmonized UAV, SDN, and NC mixture by simulating a pollution attack detection mechanism in a simplified architecture of what we proposed earlier. Overall, SDN-based UAV networks offer great potential for wireless communications which means more possible ways to enhance reliability. Our future research will focus on combining innovative technologies and creating new algorithms for a reliable 6G network while keeping it cost-effective and easy to implement.

  Fig 1 illustrates our proposition on the right side versus the existing one on the left side. More detailed explanations are explained in what follows.

Fig. 1 .

 1 Fig. 1. SDN-based UAV architecture cases.

Fig. 2 .

 2 Fig. 2. Architecture diagram.

Fig. 3 .

 3 Fig. 3. Pollution detection architecture.

Fig. 4 .

 4 Fig. 4. Diagram of the pollution attack detection sequence.

Fig. 5 .

 5 Fig. 5. NC performance Line Chart.

Fig. 6 .

 6 Fig. 6. Pollution Attack Detection.

Fig. 7 .

 7 Fig. 7. Attack detection Algorithm Evaluation.

Fig. 8 .

 8 Fig. 8. Organizational chart of the solution.