Sophie Aboughe-Angone

Eric Nguema-Ona

Thaddée Boudjeko

Azeddine Driouich

Keywords: plants, polysaccharides, pathologies, metabolic diseases, ethnobotanical

The greatest part of the world's population relies on plants as its source of medicines. The crude vegetable drugs or their extracts are utilized, primarily by lay persons either as a form of 'alternative' therapy for the treatment of diseases often of a chronic nature or to attain or maintain a state of improved health. While western developed countries have turned their efforts to promoting and developing a worldwide chemical and pharmaceutical industry, most of the southern countries, including African ones, have neglected their ethnobotanical legacy. Nowadays, the economic crisis and the high costs of most of the modern pharmaceutical based treatments have enhanced a come-back to secular and indigenous medicines. Starting from herbal teas to the development of sophisticated therapies based on plant decoctions or well-purified phytochemical compounds, plant-derived components have been proven to be effective in the prevention or treatment of skin-related pathologies, cancers, metabolic diseases such as obesity and diabetes. These secular treatments include body-washes, decoctions, massages, ingestions, etc.... The aim of this review article is to describe plant-derived cell wall polysaccharides shown to exhibit biological activities. If better characterized, studied and valorised, they may play an important role in health care policy in the world, and more particularly in numerous developing countries in the next decades.

I. INTRODUCTION The plant-derived polysaccharides

During photosynthesis, plants convert inorganic carbon into organic forms. This carbon enters several biosynthetic pathways and a broad range of primary and secondary metabolites are synthesized. More than 80% of this carbon is deposited in an extremely rich polysaccharide compartment that surrounds plant cells and is best known as the plant cell wall. Starch, the plant polysaccharide found in the cytoplasm, differs from cell wall polysaccharides in its digestibility by plants, microbes and vertebrate enzymes. The plant cell wall (sometimes called non-starch polysaccharides) consists mainly of cellulose types of linkages that bind these monosaccharides to one another, have defined pectin as the most structurally complex polymer in the wall [9]. Indeed, galacturonic acids represent sometimes over 60% of the molecular weight of pectic material, and are found mostly as α-1,4 linked galacturonic acid polymers (or homogalacturonan), often substituted with acetyl residues, or methylesterified. Homogalacturonans can also be substituted with xylosyl residues to form a xylogalacturonan. The most complex pectin structure having a homogalacturonan backbone is the rhamnogalacturonan-II (RG-II). This homogalacturonan backbone is substituted by four different side chains, referred to as chain A-D, and having different structural compositions. These side chains contain some rare sugars like apiose, aceric acid, 2-keto-3-deoxy-D-mannooctulosonic acid (or Kdo). Two RG-II monomers bind together to form an RG-II dimmer, this binding occurs via a cross-linking with a 1:2 borate-diol ester. There is another pectic component which differs slightly from the others previously described. Rhamnogalacturonan I (RG-I) contains a backbone of the repeating [4-α-D-GalacturonicAcid-1,2-α-L-Rhamnose-1] disaccharide. RG-I is believed to interrupt the homogalacturonan pectic backbone and may form with it a continuous pectic chain [10]. RG-I is most often substituted by side chains enriched with arabinose (arabinan chain), or with galactose (galactan chain). A combination of arabinan and galactan chains is sometimes present within the same acidic pectic material, as well as different arabinogalactan (AG) chains varying in the type of linkage found between their arabinosyl and galactosyl residues [11]. Type I arabinogalactan (AG-I) refers to a β-1,4 galactan chain linked to position 2 of the rhamnosyl residue of a RG-I backbone. The β-1,4 galactan chain can be substituted at O-3 by side chains containing 1,5linked L-arabinofuranose residues, whereas the type II consists of a (1→3)-linked β-D-Galp main chain, substituted at O-6 by 1,6-linked β-D-Galp side chains [12]. While AG-I is specific to pectic polysaccharides, the type II arabinogalactan (AG-II) may also be found associated to them as well as with arabinogalactan proteins (AGPs). In addition to this AG-II moiety, AGPs can also bear microfibrils, hemicelluloses and pectins [1]. Phenolic compounds and sometimes heavily glycosylated cell wall proteins which are also present in it. The structural complexity of this cell wall from lower and higher plants, as well as the recent advances in genome sequencing program of some model plant organisms (Arabidopsis thaliana, Oriza sativum, Chlamydomonas reinhardtii; respectively Alonso et al. 2003 [2]; Matsumoto et al. 2005 [3]; Merchant et al. 2007 [4], showed that more than a tenth of plant genomes code for enzymes involved in plant cell wall biogenesis. These polysaccharides and glycoconjugates fulfil different biological functions in plants including the control of cell growth and a role of barrier against pathogens. These components consist often of repetitive units of saccharides composed of various sugars including hexoses, pentoses, deoxy sugars, uronic acids.

One of these repetitive units is cellobiose, a [4-β-D-glucose-1,4-β-D-Glucose-1] disaccharide, the elementary brick of the cellulose β-1,4 glucan chain. More complex repetitive units are found with hemicellulosic compounds. These polymers include xyloglucans (substituted β-1,4 glucan chain with various neutral sugars), xylans and arabino/glucurono-/xylans (substituted β-1,4 xylan with arabinosyl or glucurunosyl residues), mannans, galacto/gluco-/mannans (substituted β-1,4 mannan/ glucomannan with galactosyl or glucuronosyl residues) and are expressed in the primary and/or secondary cell walls of various organisms starting from algae to dicotyledonous plants. For a detailed overview of the on hemicellulose structure and biosynthesis, we suggest Lerouxel et al. (2006) [5], and Moreira and Filho (2008) [6]. In vivo, hemicelluloses are believed to interlink cellulose microfibrils, building an important cellulose-hemicellulose network in the cell wall [7]. Various hemicellulose polysaccharides showing different repetitive units (also called oligosaccharides) can be found among plant species. For instance, different hepta-to nonasaccharides defines the structure of xyloglucan [8]. The cellulosehemicellulose network is embedded in a pectinrich matrix. The numbers of acidic and neutral monosaccharides found in the chemical composition of pectins, as well as the various The complement cascade is an important part of innate immune defence [19]. Its involvement contributes to inflammatory responses and enhances immunological defence reactions. Acquired immunity supplements the protection provided by innate immunity and comes into play relatively late. B and T-Lymphocytes are actively involved in acquired immunity. B-Lymphocytes are able to specifically recognize an antigen, including soluble polysaccharide derivedimmunogens, via the production of antibodies and the activation of complement. It must also be noted that both innate and acquired responses are not completely independent since complements, which group together a set of soluble or membrane-circulating factors, allow innate and acquired immunity actors to work together.

A range of polysaccharides isolated from higher plants is considered to be biological response modifiers which have been reported to enhance various immune responses like complement activation, lymphocytes proliferation and macrophages stimulation. The use of immunomodulators derived from plants is attractive because it allows the enhancement of host-derived mechanisms while not involving the use of microorganism-specific therapeutics such as antibiotics [20].

Immunological activity of pectic polysaccharides

Many naturally occurring pectic polysaccharides from plants, bacteria, lichen and fungi [21] act on human immune system. While the term "acidic polysaccharides" generally refers to pectic polysaccharides, they may be acidic or neutral since their side chains, sometimes enriched in arabinan, galactan or AG side chains, may account for 60% of the polysaccharide molecular weight and may be found free of any galacturonic backbone attachment, but linked to AGPs. In general, the term "neutral polysaccharides", is mostly associated with hemicellulosic compounds (see section II). Several studies have described the immunomodulatory and mitogenic (proliferation of B-lymphocytes) properties of pectic polysaccharides. These pectic fractions are very often badly characterized and may play a role at different levels, varying from complement smaller arabinan chains [13]. The polysaccharide moiety of these glycoproteins represents up to 90% of the molecular weight of the molecule and their protein backbone is often enriched in hydroxyproline, serine and threonine residues [14]. AGPs specifically react with the Yariv reagent [15,16]. This interaction occurs between the AG-II moiety of the proteins and glycoside residues attached to the phenylazo ring of the chemical dye [17].

The wide structural diversity of plant cell wall polysaccharides (or non starch polysaccharides) reflects the different mechanisms of action these polysaccharides may use to stop, prevent or heal various infections or pathologies and an abundance of literature has reported that some of their sub-fractions are able to activate the animal immune system and modulate obesity.

II. Immunomodulation of the immune system

Vertebrates have developed a sophisticated immune system which protects them from intruders which can be chemical molecules, pollen, food or whole microorganisms. Immunestimulating molecules can promote the immune system to an activated state which considerably increases its capacity to fight and eliminate intruders during infection [18]. Among these molecules, plant cell wall polysaccharides are shown to be nonself antigens, heavy molecular weight molecules, structurally complex components which are also partially degradable by host immune system. These criteria qualify almost all plant cell wall polysaccharides as immunogen molecules that are able to activate the vertebrate immune system at different levels, giving them a better means to react efficiently against different kinds of infections [19].

The immune system involves all the mechanisms used by the body for protection from intruders. Two responses, innate immunity and acquired immunity, can be activated for the fight. Innate immunity refers often to the body surface (skin, mucous membranes) as well as internal components including phagocytic cells like macrophages, and other soluble factors released by leucocytes [19]. The term immunomodulation refers to the activation of the immune system involving complement activation.

pectic polysaccharide isolated from both GOA1 and GOA2 fractions caused a complement system activation and B-cells proliferation, while a lower activity was associated with the RG-II pectic polysaccharide isolated from the GOA2 fractions [20]. The active extract from Vernonia kotschyana (Asteraceae) roots, another medicinal plant used in Mali for the treatment of gastrointestinal disorders and wound healing, is part of a registered improved traditional medicine called Gastrocedal [32]. The polysaccharides extracted from these roots with hot water also show some activity on the complement system. The active fraction is an RG-I polysaccharide with side chains consisting of AG-I and AG-II [27,33].

While the acidic RG-rich backbone was found to efficiently activate the complement system, the AG-II side chain it carried was shown to have a mitogenic activity.

Taking advantage of specific carbohydrate hydrolases, different researchers have tried to characterize the biologically active motifs associated to the AG-II side chains found in RG-I.

Studies on Biophytum petersianum Klotzsch (Oxalidaceae), a Malian medicinal plant used to heal various types of illnesses related to the immune system (inflammations, fever, malaria, joint pains, and wounds), showed that, the AG side chains associated to RG-I [29] exhibit an immunomodulatory activity. Potentially active fractions were characterized after enzymatic degradation using endopolygalacturonases. Partially characterized released subfractions were able to stimulate various cellular actors of the immune system [29]. Sakurai et al. (1998) [34] have shown that the biological activity (activating and anti-ulcer activities) of Bupleurum falcatum (Umbelliferae) a Sino-Japanese plant, was associated to RG core substituted with one galactosyl side chain (β-1,3galactosyl chains to which β-1,6-linked galactosyl chains) bearing α-1,5 arabinofuranosyl chains. β-1,6 linked galactosyl residues carrying one terminal Glucuronic acid (GlcA) or 1,4-methylated GlcA, are responsible for the major immunogenic activity. The biologically active AG-II fraction was called bupleuran 2IIc.

In vitro studies showed that bupleuran 2IIc induces proliferation of B-cells in the absence of macrophages, and the activated B-cells fixation to B-cells proliferative activities [START_REF] Yamada | Contribution of pectins on health care In[END_REF][START_REF] Yamada | Complement activating polysaccharides from medicinal herbs[END_REF][START_REF] Wagner | Bioactive. Carbohydrate Polymers[END_REF]25,26,27,28,20,29,30].

a) Immunological activity of acid and neutral pectic polysaccharides

Different types of acid polysaccharides such as RG-I, RG-II and homogalacturonans isolated from plants used in traditional medicine, have been identified for their activities on the complement system [START_REF] Yamada | Complement activating polysaccharides from medicinal herbs[END_REF]. They also interfere with macrophage and lymphocyte activities. Several studies have shown that pectic fractions that have an activity on the immune system are enriched of galacturonic acids and also contain often heavily branched AG-I or AG-II. The first AG isolated and found to have an immunomodulatory effect on the complement system is the AG-II extracted with hot water from the roots of Angelica acutiloba [31]. Diallo et al. (2003) [26] showed that the pectic polysaccharides isolated from the leaves of Trichilia emitica (Miliaceae), a plant used in traditional medicine in Mali, activated the complement system and induced the proliferation of T and B-lymphocytes. The fractions extracted with hot water, a treatment known to isolate mostly pectic extracts, were found to be active on complement system. These fractions contain the acidic RG-I backbone bearing an AG-II side chain which binds to the Yariv reagent. Partial acid hydrolysis with oxalic acid removes mostly the β-1,3 and β-1,5 branched β-1,3 galactosyl side chains. Biological tests prior to and after partial hydrolysis revealed that most of the activities are due to the type II arabinogalactosylated side chain of the RG-I fraction, and the removal of arabinosyl residues by oxalic acid treatment decreased this biological activity. Similar studies on Glinus oppositifolius (Aizoaceae), a plant found in western Africa, also showed the activation of complement system by two pectic polysaccharides (GOA1 and GOA2). GOA1 showed a heavily and mixed chains containing AG-I and AG-II linked to RG backbone, while GOA2 had a more classical pectic structure with a polygalacturonan backbone interrupted by RG regions, poorly substituted with AG-II [28]. Both fractions were able to activate the complement system and to induce the chemotaxis of macrophages and T-cells at the inflammation site. Further chemical analyses showed that an RG-I Europe. It is also known for its aphrodisiac properties as well as for its use in treatment of rickets, diabetes, dyspepsia, rheumatism, anaemia and constipation [START_REF]Spices, Herbs and Edible Fungi[END_REF][START_REF] Ramesh | [END_REF], some studies show that galactomannan fractions extracted from Fenugreek exhibited immunostimulatory activity [START_REF] Ramesh | [END_REF]. The Aloe vera ace mannan-rich fraction was also found to exhibit immunomodulatory activities [39]. Zha et al. (2007) [40] have purified a hemicellulosic polysaccharide (HPS-1B23) (Figure 1) from the stems of Dendrobium huoshanense, a plant used in traditional Chinese medicine as a therapeutic agent for curing cataract, throat inflammation, fever and chronic superficial gastritis or as a tonic [START_REF] Bao | Medical Dendrobii in China[END_REF]40]. HPS-1B23 stimulates the immune system at different levels (stimulation of interferon gamma (IFN-γ) and the secretion of tumour necrosis alpha factors (TNF-α). The active hemicellulosic fraction, enriched with glucose, mannose and galactose, may consist of a mixture of branched galactomannans and galactoglucomannans. Immunological activities were also found on Tamarind (Tamarindus indica) seed xyloglucans. These hemicellulosic polysaccharides, consisting of a β-1,4 glucosyl backbone substituted by xylosyl or galactoxylosyl residues have shown several biological activities such as a marked inhibitory effect on the binding of the polyomavirus family (BK) to cells, or immunomodulatory effects on the complement system [START_REF] Lanhers | [END_REF]. Tamarind seed xyloglucans may also induce the secretion of cytokines in various skincell lines [43]. Characterization and biological activity tests were recently initiated on Polysaccharides as immunomodulators of the immune system are induced into antibody-forming cells in the presence of interleukin-6 [34]. Another study on Glinus oppoditifolius, a plant used in traditional medicine in Mali for the treatment of several diseases, showed that a β-1,3 galactosyl side chain containing a terminal glucuronic acid (GlcA), or a 4-O-methyl-GlcA is responsible for the biological activity. This pectic extract binds to B-cells receptors and induces their proliferation [20].

b) Immunological activity of AGPs

AG-II are heavily branched side chains linked to RG-I pectic polysaccharides, but also to AGPs. Indeed, AG-II may be found associated to the RG-I backbone, linked to AGPs, or free in the wall (maybe cleaved by wall hydrolases in muro) and have the ability to bind the Yariv reagent [32,30]. These common chemical properties make it sometimes difficult to attributes the biological activities found to a particular molecule. [36] showed that the AGP isolated from the juice of the aerial parts of Echinacea purpurea bind to the lymphocytes, monocytes and granulocytes of different donors.

Extensive reports of studies showing the anticomplementary activity of many other pectic polysaccharides are summarised in Table 1.

Immunological activity of hemicellulosic polysaccharides

Hemicelluloses are neutral polysaccharides which account for approximately 35% of the biomass of annual and perennial plants and hemicellulosic polymers isolated from different plants used in traditional medicine. Like pectic polysaccharides, they present immunomodulatory and immunostimulating activities.

Bioactive galactomannan which are hemicellulosic polysaccharides have been described in Fenugreek (Trigonella foenum-graecum L). This is an annual leguminoseae herb used as a spice (seeds), or as vegetable (leaves). Fenugreek is an indigenous plant growing in western Asia and in the south of

Table 1.

A few examples of pectic polysaccharides with anticomplementary activity isolated from medicinal higher plants. polysaccharides from Fleurya aestuans (Urticaceae), a Gabonese medicinal plant used as an antipyretic, antirhumatic and diuretic and for wound-healing [44]. The alkaline hemicellulosic fractions were fractionated into xyloglucan-rich and acid xylanrich fractions.

Both fractions showed immunostimulatory activity on B-cells. The tests were performed on human blood cells using different concentrations for both extracted hemicellulosic subfractions (1, 10, 50 and 100 µg.mL -1 ). When treated respectively with xyloglucan-rich and acid xylan-rich fractions, in the presence of Staphylococcus aureus cowan A, we found an incorporation rate of tritiaced thymidine between 45000 and 61000, and between 45000 and 100000 in human B-cells. (Figure 2) (Aboughe-Angone et al. unpublished). Other hemicellulosic polysaccharides having immunological activity are summarised in the Table 2.

Immunological activity of sulphated polysaccharides

Several sulphated carbohydrate polymers (mostly found in lower plants like algaes) were found to be active against various viral infections including important human pathogens such as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, dengue virus and respiratory syncytial virus [45,46]. Sulphated polysaccharides are isolated from algae extracts. Their biological activities vary with the molecular size of the polysaccharide, the composition, the

Tamarindus indica Seeds

Xyloglucan -Immunomodulatory activity -Proliferation of cytokines [43,[START_REF] Lanhers | [END_REF] Trigonella foenum-graecum L Galactomannan -Immunostimulatory activity [START_REF] Ramesh | [END_REF] between CD4-expressing and human immunodeficiency virus (HIV) gp160-expressing cells that was used as a model system of infection with HIV. A sulphated fucan-containing fraction isolated from the brown seaweed Stoechospermum marginatum collected from the Arabian Sea also has an inhibitory activity on herpes simplex virus type 1 and type 2 in Vero cells, [53]. Other sulphated polysaccharides with antiviral activity isolated from many other algae extracts are summarised in Table 3.

III. Dietary fibre and metabolic diseases

Polysaccharides are able to interact with other polymeric materials, including other polysaccharides, proteins and starches, either modifying each other's properties or providing a giving synergistic effect to increase viscosity or cause gelation, phase separation and other physicochemical phenomena. In recent years, much attention has been drawn to pectic polysaccharides as they have been shown to display various pharmacological activities both in vitro and in vivo, but focus has especially been on their ability to modulate immune response [START_REF] Yamada | Complement activating polysaccharides from medicinal herbs[END_REF]27,33].

The term "dietary fibre" was introduced by Hipsley in 1953 [54] and it regroups the sulfate group position, the type of linkage and molecular geometry [47,46]. It is also shown that the sulphated polysaccharides may also exhibit anticoagulant, antithrombotic, anti-inflammatory and antitumor properties [48,49,50,46]. The antiviral activity of the sulphated polysaccharides is based on the formation of similar complexes that block the interaction of the viruses with the cells. Correlations are established between different structural parameters and antiviral activity [51]. Sulphate groups require the inhibition of anti-HIV activity inhibition and the potency increases with an increase in the degree of sulphation and the molecular weight of the polysaccharide [47,46]. Mohsen et al. (2007) [46] showed that hot water-soluble sulphated polysaccharides (SP) isolated from the brown algae Surgassium latifolium, possess an antiviral activity against HSV-1 and HAV viruses. The fractionation and characterisation of the SP-III sulphated polysaccharide enriched with glucuronic acid, mannose, glucose, xylose and fucose exhibit that antiviral activity. Lee et al. (2006) [52] also show that a specific sulphated polysaccharide called naviculan, isolated from a diatom. Navicula directa shows an antiviral activity against the types 1 and 2 herpes simplex viruses, and the influenza A virus. Naviculan also shows an inhibitory effect on cell-cell fusion increase in dietary fibre intake [63]. Recent studies have shown that obesity is characterised by a subchronic inflammatory state [64,65] that is justified by an increase in the levels of many circulatory inflammatory markers like IL-6, TNF haptoglobin [66]. In relation with the pathological consequence of obesity and the strategies of treatment, the hypothesis according to which dietary fibres could have a regulatory effect on satiety or act as a physiological obstacles to food intake and energy supply were developed.

Fibbers can likewise influence the rheological properties of the gastrointestinal content than the processes digestion and absorption [67]. They essentially carry out physical action in the stomach and small intestine. Soluble fibres like βglucanes increase viscosity contributing to slowdown the emptying of gastric contents [START_REF] Edwards | The physiological effects of dietary fiber[END_REF].

The presence of viscous polysaccharides in the small intestine affects the interaction of food with digestive enzymes, consequently reducing the speed of digestion of carbohydrates [START_REF] Dougall | [END_REF]. An increase in the viscosity of the digestive content equally slows down the absorption rate of nutrients. In another way, these fibres could have an effect on blood cholesterol. In fact, fibre has the ability to bind with bile acids and prevent its reabsorption into the liver, thus inhibiting cholesterol synthesis [70]. Arabic gum, natural carbohydrate exudates from Acacia senegal and Acacia seyal trees in Africa, rich in AGPs, is not ingested in the small intestine. It has therefore applications to reduce plasma glucose and serum cholesterol [71]. A study on five healthy human volunteers taking 25 g/day of acacia gum shows a significant reduction in total serum cholesterol concentrations [72]. It is also assumed that changes in the concentration of serum cholesterol have been related to changes in intestinal microflora [73].

In the stomach, while activating the excretion of gastric acid and digestive hormones, they increase the impression of satiety and in this way could contribute in limiting obesity [START_REF] Cummings | Short chain fatty acids[END_REF][START_REF] Dougall | [END_REF]. A relationship exists between the site of degradation and the type of polysaccharide degraded. Soluble β-glucanes and arabinoxylanes are preferentially degraded at the level of the caeco-colon. Among the functional oligosaccharides, short chain-non-digestives constituents of foodstuff. The meaning of the term was then extended to include all non-starch polysaccharides, including those obtained from plant cell walls such as pectic polysaccharides and cellulose residues that are resistant to human intestinal enzymatic digestion [START_REF] Trowell | Dietary fiber redefined[END_REF]. Dietary fibres originating from plant cell walls are mostly water-soluble and include plant exudates (gum), such as Arabic gum, Ghatti gum, Tragacanth gum and Karaya gum and mucilages produced by the epidermis of the seeds of certain species [56]. They also include soluble polysaccharides produced by microorganisms such as Gellan gum, Xanthan gum and Curdlan.

More recently, the definition has been extended to include non-digestible oligosaccharides such as fructans, including inulin and other oligofructoses, resistant starches and synthetic polymers such as polydextrose [57,58]. The American Association of Cereal Chemists adopted the following definition: "Dietary fibres" are the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human intestine with complete or partial fermentation in the large intestine" [START_REF]Dietary Fiber Technical Committee. The definition of dietary fiber[END_REF]. They promote beneficial physiological effects including the management of obesity, diabetes, laxative, and/or blood glucose attenuation.

The gastrointestinal tract is heavily populated with bacteria, mainly the strictly anaerobic ones.

Most of these organisms are benign to the host; however, certain gut species are pathogenic and may be involved in the onset of acute and chronic disorders [60]. Due to their chemical structure, the polysaccharides are substrates that can only be consumed by a limited number of bacteria, thus stimulating their growth. Among the group of bacteria present in the gastrointestinal tract, the bifidobacteria and lactobacilli are those that mostly utilize oligosaccharides, being considered as the only microorganisms able to beneficially affect the host's health [61,62].

Obesity, which is defined as an excess in weight and a body mass index superior to 30% has become in the past few years a major public health problem in our society. Obesity and obesity related type II diabetes are typical diseases of the modern Western society. Current recommendations for the management of these conditions include an biological activities and may be helpful to prevent or heal various infections or diseases. In this respect, polysaccharides are still less studied and valued, when compared to some others plant molecules like alkaloids for instance. In some The recent identification of almost all the genes involved in cell wall polysaccharide biosynthesis has increased our understanding of this process. More interestingly, resources are available among different lower and higher plant species. For instance, The CAZy database (Carbohydrate Active enZymes; www.cazy.org)F compiles all the genes from different plant organisms having a proven enzymatic activity. Understanding the molecular mechanisms underlying the biosynthesis of these components has recently boosted biotechnological research in several domains of plant research, including plant biomass derived biofuel, agriculture, etc. The opportunity could also be taken to develop biotechnological approaches to increase the availability of plant cell wall carbohydrate components showing a promising biological activity during the treatment of various diseases. Similar approaches, often coupled to organic fructo-oligosaccharides (FOS), given at the dose of 10% in the diet of rats for a few weeks reduces hepatic triglyceride content, postprandial glycemia and triglyceridemia in normal rats, lessens hepatic steatosis and fat mass development in obese Wistar rats fed with high fat diet. This phenomenon could be partly explained by a satiatogenic effect of FOS [START_REF] Delmée | [END_REF]76].

Other insoluble polysaccharides could equally be degraded in the large intestine; this is the case of linear xylanes. The kinetics is slow but the degradation is total. However, xylanes and arabinoxylanes from lignified tissues are more resistant [77]. The bacterial fermentation of certain constituents of fibres leads to the formation of many products which play an important role in the physiology of the large intestine and in metabolism [78].

Diabetes mellitus is a common chronic metabolic disorder involving carbohydrate, fat and protein metabolism and characterized by glycosuria and hyperglycemia. The treatment aims at regularizing blood glucose level by the use of hypoglycaemic agents, α-glucosydase inhibitors, sugar-restricted diets, and complex carbohydrate (fibber) preparations which delay the absorption of glucose from the gut [START_REF] Willianson | Selection, Preparation and harmacological Evaluation of Plant Material[END_REF]. Recent works suggested that complex polysaccharides could interfere with the receptor signal transduction pathway of insulin, notably with Protein Tyrosine Phosphatase (PTP1B), a potential therapeutic target of type 2 diabetes located on the cytosolic surface of the endoplasmic reticulum. Protein tyrosine phosphatases regulate the insulin signaling by mediating the balance between insulin receptor phosphorylation and dephosphorylation. Recently, Mao et al. (2007) [START_REF] Mao | [END_REF] showed that Astragalus water-soluble polysaccharides increase insulin sensitivity by decreasing the over expression of PTP1B in type 2 diabetes animal models.

GENERAL CONCLUSION

Plants remain the most abundant source of foods, phytomedicinal remedies and natural composites able to meet numerous needs of human beings. We showed here that plant-derived cell wall polysaccharides display a wide variety of structure. Several of these molecules have chemistry synthesis, have been followed before in the past to produce other plant-derived metabolites at large scale. In contrast to other plants metabolites, the structural complexity does not easily allow for the development of an organic chemistry-based approach. Only a large scale production of these active carbohydrates can meet the needs of a numerous population and the development of phytochemical pharmaceutical industry. The extensive screening of potential and uncharacterized bioactive polysaccharides from southern countries, along with the ethnobotanical and ethnopharmacological legacy, their complete purification and chemical characterization, and the availability of biotechnological resources may allow us to write a new story on plant-based medicinal therapy.

Figure 1 .

 1 Figure 1. Proposed structure of repeat unit of HPS-1B23 from Dendrobium huoshanense as proposed by Zha et al. 2007. Legend: : α-D-Glup, : OAc, : α-D-Manp, : α-D-Gal

  Classen et al. (2006) [35] showed the immunomodulatory activity of the AGPs of Baptisia and Echinacea species. These AGPs have an influence on proliferation and the production of immunoglobulin M (IgM) of lymphocytes in mice, the stimulation of nitrite and the production of interleukin-6 by macrophages. Thude et al. (2006)

Figure 2 .

 2 Figure 2. Schema of the proliferation B-cells induces by the hemicellulosic polysaccharides rich fraction xyloglucans (F. XyG) and rich fraction acidic xylans (F. Xylans).

Table 1

 1

continued..

Table 2 .

 2 Example of hemicellulosic polysaccharides with immunological activity isolated of a few medicinal plants.

	Botanical name and part used	Polymers type	Activities	References
	Aloe Vera	Acemannan	-Immunomodulatory activity [39]
	Echinacea purpurea	Xyloglucan	-Stimulation of macrophages	[96]
	Aerial parts		and production of
			interleukine-1
	Ganoderma lucidum	β-Glucan	-Induce tumour necrosis	[97]
	Libyan dates (Phoenix dactylifera L.)	β-Glucan	-Anti-cancer	[98]
	Fruits
	Plantago asiatica	Glucurono-arabinoxylane -Complement System	[31, 99, 100]
	Seeds
	Plantago major	Heteroxylan	-Complement System	[101]
	Seeds
	Poria cocos sclerotium	β-Glucan	-Anti-tumour	[102]
	Entire plant
	Rhynchelytrum repens (Wild)	β-Glucan	-Hypoglycaemic activity	[103]
	Entire plant

Table 3 .

 3 Some sulfated polysaccharides with antiviral activity.

	Botanical name	Polymers type	Activities	References
	Agardhiela tenera	Sulfated Galactan	VIH1-2 and HSV1-2	[47]
	Gayralia oxysperma	Sulfated heterorhamnan herpes simplex virus	[104]
			(HSV)
	Gigartina aciculaire	Carrageenan	VIH1-2 and HSV1-2	[47]
	and Gigartina pistillata
	Navicula directa	Sulfated Polysaccharide	VIH-1 and HSV1-2	[52]
		(Naviculan)
	Nothogenia fastigiata	Xylomannane	VIH1-2 and HSV1-2	[47]
	Porphyridium sp.	Sulfated Polysaccharide HSV1-2	[105]
	Sargassum alga	Sulfated Polysaccharide Herpes simplex virus	[106]
			(HSV1-2)
	Scinaia hatei	Sulfated xylomannan	HSV	[107]
	Stoechospermum	Sulfated fucans	HSV1-2	[53]
	marginatum


Olfa Douchiche

Odile Soret-Morvan

Wided Chaïbi

Claudine Morvan

Florence Paynel

Characteristics of cadmium tolerance in 'Hermes' flax seedlings: Contribution of cell walls

Introduction

Cadmium is a major environmental pollutant, with concentrations that can reach 200 mg kg À1 in soil (e.g. [START_REF] Dahmani-Muller | Strategies of heavy metal uptake by three plant species growing near a metal smelter[END_REF]. At the whole-plant level, the major symptom of Cd toxicity is a global decrease in growth rate [START_REF] An | Soil ecotoxicity assessment using cadmium sensitive plants[END_REF]. At the organ level, browning and enlargement of roots are often reported [START_REF] Barceló | Plant water relations as affected by heavy metal stress: a review[END_REF], while in leaves, chlorosis, stunting, and rolling are often observed (Sanità di [START_REF] Di Toppi | Response to cadmium in higher plants[END_REF]. Within cells, Cd toxicity is associated with oxidative stress [START_REF] Sandalio | Cadmium-induced changes in the growth and oxidative metabolism of pea plants[END_REF], membrane damage, and alteration of numerous enzymatic activities [START_REF] Békésiová | Heavymetal stress induced accumulation of chitinase isoforms in plants[END_REF]. Some plant species exhibit resistance or tolerance to heavy metals, in varying degrees [START_REF] Clemens | Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants[END_REF]. One resistance strategy is to avoid metal absorption by the roots. The avoidance mechanisms include reduction of absorbant hair surface and secretion of organic acids or mucilages into the rhizosphere. Another resistance strategy is cation compartmentalization in the root either by restricting them in cortical tissues, or by decreasing the vessel number and diameter. Within the cell, several strategies are used to maintain cellular metabolism such as exclusion at the surface of plasmalemma, sequestration in vacuole, complexation via metal-chelating molecules in cytosol and immobilization in cell wall (CW) [START_REF] Clemens | A long way ahead: understanding and engineering plant metal accumulation[END_REF]. Indeed, the CW is the first barrier to toxic metals in the environment [START_REF] Wang | Subcellular distribution and chemical forms of cadmium in Bechmeria nivea (L) Gaud[END_REF]. Immobilization of Cd in the CW of maize [START_REF] Lozano-Rodriguez | Distribution of cadmium in shoot and root tissues of maize and pea plants: physiological disturbances[END_REF] and lettuce [START_REF] Ramos | Cadmium uptake and subcellular distribution in plants of Lactuca sp. Cd-Mn interaction[END_REF] has been demonstrated to contribute to Cd tolerance in these species.

Flax can accumulate high levels of Cd and is considered as a tolerant species. [START_REF] Shi | Cadmium tolerance and accumulation in eight potential energy crops[END_REF] reported that flax plants cultivated in the presence of 50-200 mg Cd kg À1 soil exhibited a limited reduction of growth. The authors concluded that flax has an innate resistance to Cd stress and is moderately tolerant (tolerance index in the range of 40-62%).

In our laboratory growth conditions, we also observed that 'Hermes' seedlings could tolerate high concentrations of Cd, up to 0.5 mM, with little variation of the mass of the hypocotyl [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF]. At this concentration, the organization of the seedling tissues is unaffected by Cd, however, the metal provokes a reorientation of the hypocotyl growth axis, i.e. a reduction in elongation and an enhancement of cell expansion. These structural alterations are associated with a significant increase of the thickness of the cell walls, accompanied by a variation in uronic acids originating from pectin remodelling and an increase in their cross-linking. Similar to our previous report [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF], the present study was conducted with 0.5 mM Cd, but at 10 and 18 days, in order to investigate the growth response, the Cd content and the level of antioxidative activities of all organs. These two growth stages are representative of the development of primary tissues (10 d) and tissues differentiating secondary wall ⇑ Corresponding author. Tel.: +33 235146751; fax: +33 235146615. E-mail address: florence.paynel@univ-rouen.fr (F. Paynel).

1

(18 d). Particular attention was paid to pectin content (through the level of uronic acid) and the activity of CW remodelling enzymes. Altogether, the data provide new insights into mechanisms of Cd tolerance.

Materials and methods

Plant material and growth conditions

Seeds of flax (cv 'Hermes', a gift of the Cooperative Terre de Lin, France) were sown on culture medium containing either 2.6 mM Ca(NO 3 ) 2 or 0.5 mM Cd(NO 3 ) 2 and 2.1 mM Ca(NO 3 ) 2 , as previously reported [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF]. Roots, hypocotyls and epicotyls (including cotyledons) were excised from plantlets collected after 10 or 18 d (10 d and 18 d respectively). All plantlets whose hypocotyl length was less than 1 cm were not collected to avoid analysing plants experiencing extreme toxicity. Three independent culture experiments were run.

Histological study

Hand cross-sections of root were stained with carmine-green as previously described [START_REF] Brutch | Characters of fibre quality in lines of flax genetic collection[END_REF] and observed with a light microscope (Leica microsystem, Austria).

Cadmium and cation contents

After digestion of dried samples with HNO 3 /HClO 4 (3/1; v/v), the content of Cd and cations (Ca, Mg, Na, K, Mn, Fe) in the organs were measured by atomic absorption spectrometry (Analyst 300, Perkin-Elmer, USA).

Antioxidative enzyme activities

Fresh tissue was ground in 100 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 1% (w/v) polyvinylpyrrolidone and 1 mM phenylmethanesulfonyl fluoride. Homogenate was centrifuged (12 000g, 20 min, 4 °C) for protein [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] and enzyme activity measurements.

Activities of superoxide dismutase (SOD, E.C. 1.15.1.1), catalase (CAT,E.C. 1.11.1.6) and ascorbate peroxidase (APX,E.C. 1.11.1.11) were assayed as described by [START_REF] Qiu | Antioxidative response to Cd in a newly discovered cadmium hyperaccumulator, Arabis paniculata F[END_REF]. Guaiacol peroxidase (GPX, E.C. 1.11.1.7) activity was measured as described by [START_REF] Paynel | Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl[END_REF]. Activities were expressed as nkatal g À1 FW, except for SOD (U g À1 FW).

Lipid peroxidation and protein-bound carbonyl content

Lipid peroxidation was determined by measuring the concentration of thiobarbituric acid-reacting substances (TBARS) as described by [START_REF] Buege | Microsomal lipid peroxidation[END_REF] and was expressed as nmol TBARS g À1 FW.

Carbonyl groups were assayed using the dinitrophenyl hydrazine method, according to [START_REF] Levine | Carbonyl assays for determination of oxidatively modified proteins[END_REF]. Carbonyl content was calculated using an extinction coefficient for aliphatic hydrazones of 22 000 M À1 cm À1 and was expressed as nmol g À1 FW.

Extraction of cell-wall uronic acids

Fresh tissue was ground in liquid nitrogen. The powder was treated in organic solvents for the isolation of CW. The CW yield was the mass (g) of dry CW per 100 g of fresh organ. Then, the CW was successively treated with trifluoroacetic acid (2 M TFA, 110 °C 2 h) and sulfuric acid (concentrated H 2 SO 4 ,2 5°C 1 h, followed by a dilution to 1 M, 110 °C 2 h) [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF].

The two filtrates (TFA and H 2 SO 4 ) were kept aside for uronic acid (UA) analyses [START_REF] Blumenkrantz | A new method for quantitative determination of uronic acids[END_REF].

Cell-wall remodelling activities

Fresh tissue was ground in liquid nitrogen, and the ionicallybound proteins were obtained as previously described [START_REF] Paynel | Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl[END_REF]: CW pectin methylesterase (CW PME, E.C. 3.1.1.11) activity was measured using the alcohol oxidase method and CW peroxidase (CW GPX, E.C. 1.11.1.7) activity was determined using guaiacol as substrate.

Results

Impact of cadmium on growth and anatomy

The elongation of flax plantlets, cultivated on 0.5 mM Cd was significantly reduced, especially in the root (Fig. 1A). The biomass and the mass ratio between root and shoot were moderately affected, mainly because the growth axis was reoriented. The mass tolerance-index (TI = 100 Â [biomass in Cd]/[biomass in control]) was high, in the range of 79-84%. Also, the water content of all organs remained constant.

Cd did not have an impact on root histological organization, but the root diameter was increased mainly due to the bigger cortical cells (Fig. 1B). More secondary roots were counted per section, mainly due to the length reduction of the primary root. The carmine-green staining of the rhizodermis, endodermis and xylem appeared more contrasted. Xylem was more expanded and exhibited four poles (Fig. 1B4) compared to the two poles in control roots (Fig. 1B3). The expansion of the xylem into four poles started as soon as 10 d (data not shown). This would correspond to an acceleration of the differentiation of tissues with secondary wall.

Cadmium accumulation and its effect on cation content

In treated plantlets, Cd concentration was about four times higher in roots than in shoots and was equivalent in hypocotyl and epicotyl (Table 1). Cd content in roots increased with time but did not vary much in shoots. When expressed as amount per plant, 3.2 lg of Cd was accumulated in root compared to 1.7 lg in shoot, at 18 d.

Among the ions studied, Na and K were the main ones accumulated in all organs. After 18 d of treatment, the concentration of Na decreased specifically in root while that of Mg increased in shoot. Cd also impacted other micronutrients, with a marked and opposite variation of Fe (decrease) and Mn (increase) content in all organs.

Effect of cadmium on lipid peroxidation, carbonyl content and antioxidative enzyme activities

Cd treatment had only a limited effect on lipid peroxidation and carbonylation of proteins. TBARS content increased only in 10 d Cd-exposed root and in 18 d Cd-exposed epicotyl (Fig. 2A). No significant difference in soluble protein level was observed between treated and control organs, except for an increase in protein observed in 18 d Cd-treated roots (Fig. 2B). Within these organs, the amount of oxidatively modified amino acids accounted for only about 10 ppm (Fig. 2C).

The impact of Cd on the activity of antioxidative enzymes (i.e. SOD, CAT, APX and GPX, Fig. 2D-G) was insignificant in most organs. At 10 d, only root APX displayed a significantly higher activity level than control. At 18 d, only CAT activity was significantly increased in Cd-treated roots, while in treated epicotyls SOD activity decreased and GPX activity increased. The GPX activity possibly compensated for the observed SOD decrease.

Altogether, the variations of lipid peroxidation between organs in presence and absence of Cd were correlated to those of APX activity (r 2 = 0.94).

Impact of cadmium treatment on the cell-wall yield and uronic acid content

To assess the effect of Cd on pectin metabolism, we analysed the CW yield and CW-UA content (Table 2). At 10 d, CW yield increased in root and hypocotyl following Cd treatment. This increase resembled the age-dependent CW yield increase observed in untreated tissues and might be related to the acceleration of xylem differentiation described above (see Fig. 1).

Concerning UA, they were assayed firstly in TFA (hydrolysing matrix polysaccharides) and designated as TFA-UA, and secondly in H 2 SO 4 (hydrolysing the residual sugars) and named H 2 SO 4 -UA. According to [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF], TFA-UA were more extractable and consisted of rhamnogalacturonan (RG) structure while H 2 SO 4 -UA would instead have originated from homogalacturonan (HGA) that was either associated 10 d -Cd + Cd Root length (cm) 5.55 ± 0.26 3.03 ± 0.24** Shoot length (cm) 3.68 ± 0.13 2.86 ± 0.19** Root FW (mg plant -1 ) 16.1 ± 0.8 12.9 ± 1.3* Shoot FW (mg plant -1 ) 37.6 ± 1.5 31.9 ± 1.9* Water content (%)

92.1 ± 0.9 92.5 ± 0.5 18 d -Cd + Cd Root length (cm) 8.17 ± 0.65 3.69 ± 036** Shoot length (cm) 5.53 ± 0.23 4.23 ± 0.21** Root FW (mg plant -1 ) 16.3 ± 0.9 13.0 ± 1.2 Shoot FW (mg plant -1 ) 42.6 ± 1.5 34. with cellulose or precipitated beside cellulose during TFA treatment.

The UA amount released in TFA was generally larger than in H 2 SO 4 (except in Cd-treated hypocotyl), indicating a high level of

Root Hypocotyl Epicotyl

Carbonyl content of proteins Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). Values represented mean ± SE (n = 3). Ã and ÃÃ indicated significant differences between ÀCd and +Cd, at p < 0.05 and p < 0.01, respectively.

Table 2

Effect of cadmium on the cell-wall yield and on the extractability of uronic acids. Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). Cell-wall (CW) yield was the mass (g) of dry CW per 100 g of fresh organ. Uronic acids (UA) were extracted by TFA, followedb yH 2 SO 4 and expressed as mg g À1 CW dry weight. CW yield was the average of three experiments (SE < 10% RG in CW matrix. In 10 d treated plantlets, the TFA-UA level decreased whereas the UA amount released in H 2 SO 4 was enhanced.

At 18 d, the main Cd impact led to an increase of TFA-UA. Globally, the total UA amount (TFA + H 2 SO 4 ) was not affected by Cd, but increased with age from 135 ± 12 to 160 ± 10 mg g À1 CW. Such values corresponded to a maximum cation exchange capacity (CEC) of 675-800 leq g À1 CW. These CEC values would be high enough to neutralize the moderate absorbed amount of Cd (estimated for 12-60 leq g À1 CW).

Effect of cadmium on cell-wall pectin methylesterase and peroxidase

The effect of Cd on CW PME activity was maximal in hypocotyl with an increase of 60% and 67% at 10 d and 18 d, respectively (Fig. 3A). In root, the CW PME activity decreased slightly at 10 d while it increased at 18 d. In epicotyl, only a statistically unsignificant increase of PME activity was observed at 18 d.

Compared to CW PME activity, the effect of Cd on CW GPX activity was observed later (at 18 d), with an increased activity in both root and hypocotyl (Fig. 3B). These data suggested that the crosslinking of pectins described in hypocotyl [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF] might also occur in root. Although the CW GPX activity increased with age in epicotyl, no impact of Cd was observed whatever the age.

Discussion

Flax 'Hermes' is a variety tolerant to cadmium

Plant growth inhibition is commonly used as an indicator of Cd toxicity. In flax, the main impact of Cd treatment was reorientation of the growth axis of the hypocotyl [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF] and of the root (the present study).The tolerance index was in agreement with a recent study that demonstrated the Cd tolerance capacity (59% < TI < 72%) of another flax cultivar [START_REF] Shi | Cadmium tolerance and accumulation in eight potential energy crops[END_REF]. Moreover, Cd had no visible impact on organ anatomy. Hence, based on plant growth response, 'Hermes' seedlings appeared tolerant to high Cd concentration. Because the CW contributes greatly to the determination of the growth axis of organs, we discuss next whether CW yield and UA content might be related to Cd tolerance.

Some studies have shown that the CW is subject to structural modulations following abiotic stress (e.g. [START_REF] Mccann | Changes in pectin structure and localization during the growth and NaCl-adapted tobacco cells[END_REF]. Our data showed that at 10 d, Cd induced an increase of H 2 SO 4 -UA in all organs. We suggest that cells, in order to adapt the plant to Cd stress, have reinforced the cell junctions via HGA acidification and subsequently via an increase of calcium pectates. These moities would not be hydrolysed by TFA but would be detected in H 2 SO 4 . Such an hypothesis has been validated in hypocotyl where the HGA acidification was shown to be catalysed by a specific CW-PME isoform named B2, which was only present at 10 d [START_REF] Paynel | Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl[END_REF]. Also, this acidification was related with the increase of HGA immunogoldlabelling in cell junctions [START_REF] Douchiche | Spatial regulation of cell-wall structure in response to heavy metal stress: cadmium alteration of the methyl-esterification pattern of homogalacturonans[END_REF]. In root and epicotyl, such an increase of B2 would be accompanied by a decrease of one or more forms. Incidentally, the activity decrease in root could be correlated to the decrease of TFA-UA.

At 18 d, the Cd-induced increase of matrix-UA was concomitant with an increase of CW-PME and most importantly of CW-GPX activities. We suggest that, after the reinforcement of cell junctions at 10 d, cells would increase the cross-linking of the pectic matrix within the walls. This would allow an increased resistance to cell swelling observed in both root and hypocotyl. To our knowledge, only a few papers (e.g. [START_REF] Konno | Lygodium japonicum fern accumulates copper in the cell wall pectin[END_REF] have reported an impact of metal on pectin cross-linking.

Other general indicators of metal tolerance include cation absorption and antioxidative capacity. Cd had been shown to interfere with uptake, transport and use of several ions [START_REF] Das | Studies on cadmium toxicity in plants: a review[END_REF]. Our investigations showed that Cd did not have a general negative effect on cation composition, possibly because of the observed acceleration of xylem differentiation in the root and the keeping of the water uptake. However, the inhibition of Fe absorption we observed suggested that Cd might be carried into cells by Fe transporters. Although this inhibition provoked the reduction of Fe in shoots, we did not observe damages on the leaves which might be related to the enhancement of Mn observed in all organs. In stressed plants, chlorosis of leaves is among the first symptoms of Cd toxicity, resulting from the deficiency of Fe [START_REF] Siedleska | Cd/Fe interaction in higher plants: its consequences for the photosynthetic apparatus[END_REF] and/or Mn [START_REF] Goldbold | Effect of zinc, cadmium and mercury on root elongation of P. abies (Karst.) seedlings and the significance of these metals to forest die-back[END_REF]. [START_REF] Baszynski | Photosynthetic activities of cadmium-treated tomato plants[END_REF] demonstrated a partial restoration of tomato chloroplast structure damaged by Cd when Mn was added. So, in 'Hermes' seedlings, the Fe decrease seemed to be compensated by the Mn increase. Together with the increase of Mg occurring in the shoot, our data suggest no or low impact of Cd on photosynthesis, as reported by [START_REF] Shi | Cadmium tolerance and accumulation in eight potential energy crops[END_REF] who showed no effect of Cd on chlorophyll content of flax.

Measurements of carbonyl group and TBARS content allow to assess protein oxidation and lipid peroxidation and consequently to estimate oxidative stress. Increase of TBARS content in Cdtreated plants is often observed, even in hyperaccumulators [START_REF] Qiu | Antioxidative response to Cd in a newly discovered cadmium hyperaccumulator, Arabis paniculata F[END_REF]. In 'Hermes' seedlings, lipid peroxidation took place transiently in root and epicotyl while some increase of protein oxidation occurred only in root. This indicated that Cd did not induce severe oxidative stress in 'Hermes' plantlets. Moreover, the slight increase of soluble proteins observed in root was an indicator that senescence was not induced [START_REF] Costa | Influence of cadmium on soluble carbohydrates, free amino acids, protein content of in vitro cultured Lupinus albus[END_REF].

Reactive oxygen species generated by Cd are counteracted by antioxidative enzymes such as SOD, CAT, APX and GPX. The significant variations of the enzyme activities only happened when lipid peroxidation or protein carbonylation occurred. In root, our results indicated that APX and CAT functioned successively as the main H 2 O 2 quenchers. In epicotyl, the main quencher was GPX. Thus, complementary antioxidative mechanisms are used depending on the organ and the age, as reported in Cd-treated Arabis paniculata [START_REF] Qiu | Antioxidative response to Cd in a newly discovered cadmium hyperaccumulator, Arabis paniculata F[END_REF].

4.2. Tolerance of 'Hermes' plantlets would be related to cadmium compartmentalization strategy

Compartmentalization of Cd in roots contributes to tolerance at the whole-plant level [START_REF] Ernst | Metal tolerance in plants[END_REF]. In 'Hermes' seedlings, Cd was accumulated more in roots than in shoots, which is in agreement with previous reports for other flax varieties [START_REF] Angelova | Bio-accumulation and distribution of heavy metals in fibre crops (flax, cotton, hemp)[END_REF][START_REF] Shi | Cadmium tolerance and accumulation in eight potential energy crops[END_REF]. As suggested by [START_REF] Clemens | A long way ahead: understanding and engineering plant metal accumulation[END_REF], Cd compartmentalization in 'Hermes' root may be related to the enhanced suberization and lignification observed in the endodermis that might act as a barrier against the diffusion of Cd and translocation to the shoots. Also, we observed that cells (mainly cortical ones) became voluminous in root suggesting that vacuolation might increase to sequester the metal [START_REF] Verbruggen | Molecular mechanisms of metal hyperaccumulation in plants[END_REF].

In addition, tolerance may also depend on compartmentalization within the CW, although this mechanism remains controversial. Many studies on Al toxicity have shown that Al resistance is correlated with low binding to the CW [START_REF] Eticha | Cell-wall pectin and its degree of methylation in the maize root-apex: significance for genotypic differences in aluminium resistance[END_REF]. These authors found that a low pectin content and/or a high degree of methylesterification of pectin characterised the roots of Alresistance genotypes. Thus, exclusion mechanism plays a major role in Al-resistance. In contrast, other studies have described the key role of the CW in Cd neutralization and hence in Cd tolerance in plants [START_REF] Uraguchi | Contributions of apoplasmic cadmium accumulation, antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium accumulating cultivar of black oat (Avena strigosa) Schreb[END_REF], including hyperaccumulators [START_REF] Cosio | Distribution of cadmium in leaves of Thlaspi caerulescens[END_REF]. In roots and leaves of white lupin for example, the amount of Cd fixed by CW would be twice more than the amount of Cd complexed with thiol-compounds [START_REF] Vázquez | Assessing the relative contributions of phytochelatins and the cell wall to cadmium resistance in white lupin[END_REF]. Thus, the capacity of CW to bind Cd represents a protective action against the deleterious effect of this metal by reducing its cytosolic amount.

None of the previous studies of flax had highlighted the role of the CW in the fixation of Cd. Preliminary data on 'Hermes' seedlings indicated that a large amount of Cd accumulated in organs was retained in the CW (data not shown). Although artefactual adsorption of cations onto the negative charges of CW UA may occur, these data suggested that the CW could contribute to reduced entrance of Cd into the cytosol. Importantly, the present study indicates that the root CW-CEC was large enough to neutralize the totality of the absorbed Cd. This is also interesting in light of our previous studies of flax hypocotyl where we demonstrated [START_REF] Douchiche | Cadmiuminduced alteration of the structural features of pectins in flax hypocotyl[END_REF][START_REF] Douchiche | Spatial regulation of cell-wall structure in response to heavy metal stress: cadmium alteration of the methyl-esterification pattern of homogalacturonans[END_REF] a compartmentalization of lowmethylesterified HGA in the outer domain of CW that would keep Cd away from the vicinity of the plasmalemma.

Conclusion

Our study demonstrates the Cd tolerance of 'Hermes' seedlings: Cd has little effect on biomass, tissue organization, antioxidative enzymes and cation distribution. 'Hermes' tolerance was related to an adaptation of growth and a reinforcement of tissue cohesion via CW-pectin cross-linking. Interestingly, the main CW characteristics described in the stem were also observed in the root. To tolerate Cd, this variety developed a root compartmentalization strategy, and metal sequestration into CW that has to be further investigated by the localization of Cd.

Our data also showed that the shoot Cd concentration (660 mg Cd kg À1 DW) was significantly higher than the threshold (100 mg Cd kg À1 DW) defined by [START_REF] Baker | Metal hyperaccumulator plants: a review of the ecology and physiology of a biochemical resource for phytoremediation of metal-polluted soils[END_REF] for Cd hyperaccumulators, underlining the possible use of 'Hermes' in phytoremediation. However, to conclude on the phytoremediation capacity of 'Hermes', experiments have to be conducted on mature plants grown in polluted field, as described by [START_REF] Dahmani-Muller | Strategies of heavy metal uptake by three plant species growing near a metal smelter[END_REF].

Fig. 1 .

 1 Fig. 1. Effect of cadmium on the growth of flax plantlets (A) and root anatomy (B). Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). Values represented means ± SE (n = 30). Ã and ÃÃ indicated significant differences between ÀCd and +Cd, at p < 0.05 and p < 0.01, respectively. Microscopic observations of hand-cut sections were carried out on root proximal parts of 18 d seedlings, after coloration with carmine-green. B1 and B3: in the absence of Cd, B2 and B4: in the presence of Cd. B1 and B2: whole root section; B3 and B4: zoom on the central cylinder area. CC: central cylinder; Cortex: rhizodermis and cortical parenchyma; End: endodermis; FW: fresh weight, P: pericycle; Ph: phloem; RH: root hairs; SR: secondary root; X: xylem. Arrowheads: sclerified fibres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .

 2 Fig.2. Effect of cadmium on lipid peroxidation (A), soluble protein content (B), carbonyl content of proteins (C), SOD (D), CAT (E), GPX (F) and APX (G) in vitro activities. Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). Values represented mean ± SE (n = 3). Ã and ÃÃ indicated significant differences between ÀCd and +Cd, at p < 0.05 and p < 0.01, respectively.

Fig. 3 .

 3 Fig.3. Effect of cadmium on cell-wall ionically-bound pectin methylesterase (CW PME) (A) and guaiacol peroxidase (CW GPX) (B) in vitro activities. Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). Values represented mean ± SE (n = 3). Ã and ÃÃ indicated significant differences between ÀCd and +Cd, at p < 0.05 and p < 0.01, respectively.

Table 1

 1 Effect

	10 d

of cadmium on the cation content. Seedlings were grown in the absence (À) or presence (+) of cadmium and harvested at 10 and 18 d (10 d and 18 d, respectively). The results were expressed as lmol g À1 dry weight (DW). E: epicotyl; H: hypocotyl; R: root. Values represented means of three replicates (SE 6 15%).

  ). UA values represented means of five independent CW samples (SE 6 10%).

		10 d						18 d
		ÀCd			+Cd			ÀCd			+Cd
		R	HE	R	HE	R	HE	R	HE
	CW yield (%)	4.1	3.4	5.5	5.6	3.9	5.4	4.8	4.2	4.5	5.6	4.2	5.2
	UA (TFA + H 2 SO 4 )	130	148	110	121	170	117	147	184	145	157	182	141
	UA-TFA	84	74	77	68	75	59	83	81	94	104	95	83
	UA-H 2 SO 4	46	74	33	53	95	58	64	102	51	52	86	58

Acknowledgment

We thank Dr Michael Deyholos, University of Alberta (Canada) for reading the manuscript and for helpful discussion.