Biological activity exerted by oligogalacturonides with respect to the degree of polymerization
Plant cell walls represent the most abundant renewable resource on this planet. They are rich in mixed complex and simple biopolymers, which has opened the door to the development of wide applications in different technologic fields. In this regard the polymerization processes that allow the synthesis of the cell wall and their components in living models are relevant, as well as the properties of the polymers and their derivatives. Therefore this chapter outlines the basis of polymerization with a biological approach in the plant cell wall, highlighting the biological effects of plant cell wall derivatives and their current applications.
Plant cell wall is a dynamic network highly organized which changes throughout the life of the cell. The new primary cell wall is born in the cell during cell division and rapidly increases in surface area during cell expansion. The middle lamella forms the interface between the primary walls of neighboring cells. Finally, at differentiation, many cells elaborate with the primary wall a secondary cell wall, building a complex structure uniquely suited to the function of the cell. The functions of the plant cell wall may be grouped by its contribution to the structural integrity supporting the cell membrane, sense extracellular information and mediate signaling processes [ 1]. The main components of the plant cell wall involve different polymers including polysaccharides, proteins, aromatic substances, and also water and ions. Particularly, the different biomechanical properties of the plant cell wall are mainly defined by the content of the polymers cellulose, hemicelulloses and pectins and their interactions [ 2].
The rapid progress on plant cell wall research has allowed the comprehension of the different structures, their biosynthesis and functions. Nevertheless, there is a new prominent and worth line of research, the biological activity of some molecules derived from the primary cell wall polysaccharides. These active molecules named “oligosaccharins” by Albersheim in the mid 70s, include the biologically active oligosaccharides that are produced by partial hydrolysis of polymers of the cell wall. The main biologically active components of the cell wall are the pectin-derived oligosaccharides, and the hemicellulosic-derived oligosaccharides. The biological responses of plants to oligosaccharins can be divided into two broad categories: as modulators of plant defense, and plant growth and development.
This information has permitted the use of oligosaccharins as an alternative to improve different aspects such as yield and fruit quality, and may reach a higher impact in the study of the resistance of vegetable crops.
2. Plant cell wall polymers
Plant cell wall is a complex matrix of polysaccharides that provides support and strength essential for plant cell survival. Properties conferred by the cell wall are crucial to the form and function of plants. The main functions of the cell wall comprise the confer of resistance, rigidity and protection to the cell against different biotic or abiotic stresses, but still allowing nutrients, gases and various intercellular signals to reach the plasma membrane. The wall provides enough rigidity to support the heavy weight of high trees as large as 100 m height, but also is flexible and elastic allowing growth during expansion and differentiation. During growth, cell turgor pressure provides high tensile stress to the wall, enabling its enlargement due to the accumulation of polymers during a combination of stress relaxation cycles. The primary cell wall surrounds and protects the inner cell; it lies down the middle lamella during growth and expansion [ 2]. The primary wall is thought to contribute to the wall structural integrity, cell adhesion, and signal transduction. In this chapter we focus on the primary cell walls because it has been noted that most of their derivatives exert a biological function.
Plant cell wall is a dynamic and highly specialized network formed by a heterogeneous mixture of cellulose, hemicelluloses and pectins, and in some extent proteins and phenolic compounds. Wall composition in vascular plants is approximately 30% cellulose, 30% hemicellulose and 35% of pectin, with certain 1-5% structural proteins on dry weight basis. Cellulose and hemicelluloses polymers bring rigidity to the wall and pectin provides fluidity throw the gelatinous polysaccharides matrix. Cellulose and hemicelluloses are embedded in the amorphous pectin polymers and stabilized by proteins and phenolic compounds. Hemicelluloses bind to the surface of cellulose network preventing direct contact among microfibrils, and pectin are linked to hemicelluloses forming a gel phase.
3. Components and function of the primary constituents of plant cell wall
Cellulose is the main cell wall polymer that brings support to the plant. Cellulose is a linear insoluble unbranched polymer of β-(1,4)-D-Glucose residues associated with other cellulose chains by hydrogen bonding and Van der Waals forces. Cellulose chains aggregate together to form microfibrils, which are highly crystalline and insoluble structures, each one about 3 nm in diameter, chemically stable and resistant to enzymatic attack. Cellulose microfibrils comprise the core of the plant cell wall; one third of the total mass of wall is cellulose. The variation of dry weight of cellulose in a dicot such as
Microfibrils comprise two types of cellulose called cellulose Iα and Iβ. The Iα has a single-chain triclinic unit cell, whereas cellulose Iβ has two chain monoclinic unit cell. In both forms cellulose in parallel and the terminal glucose residues rotated 180° forming a flat ribbon in which cellobiose (two glucose molecules linked by a β-(1,4) bond) is the repeating unit [ 4]. Cellulose chains may align in parallel (Type I) or antiparallel (Type II) orientation to each other. Only the Type I conformation is known to naturally occur in plants; however, concentrated alkaline treatments may cause Type II cellulose to form during harsh extraction procedures. The cellulose chains may form the Type Iα or Type Iβ conformation depending on the extent of staggering of the chains in relation to each other. Probably the interaction of cellulose microﬁbrils with hemicelluloses may affect the ratio of Type Iα to Type Iβ cellulose [ 5]. The microfibrilar disposition allows the existence of micro spaces between the microfibrils that are fulfilled by matricial polysaccharides according to the age and tissue type.
Hemicelluloses are low molecular weight polysaccharides associated in plant cell walls with lignin and cellulose. These heterogenous group of polysaccharides that have β-(1,4)-linked backbones with an equatorial configuration at C1 and C4 and hence the backbones have structural similarity [ 6]. Hemicelluloses in dicotyledonous plants comprise xyloglucans, xilans, mannans and glucomannans, while the β-(1,3;1,4)-glucans are restricted to Poales and a few other groups. In addition, arabinoxylans are the main hemicellulosic polisaccharides in graminaceous species such as wheat and barley, and in grasses [ 7].
Xyloglucan (XyG) is the most abundant hemicellulose in primary cell walls found in every land plant species that has been analyzed. XyG are branched with α-D-xylose linked to C-6 of the backbone. The most frequently xyloglucan structure in dicotyledonous flowering plants is the repeating heptamer integrated by four glucans residues with α-D-xylose substituents in three constitutive glucans of the backbone, followed by a single unsubstituted glucan residue ( Figure 1). The presence of this repeating heptamer block is an indicator of the presence of XyG polysaccharides in dicots species [ 8]. Beside the XyG residues, it may contain β-D-galactose and in less proportion L-fucose-α-(1,2)-D-galactose; in all cases the galactose residues are acetylated. The fact that all the substituents of xyloglucans are conserved denotes a highly biosynthesis control. On the other hand, in graminaceous monocots, XyG consist of 1 or 2 adjacent α-(1-6)-linked xylose residues with approximately 3 unsubstituted β-(1-4)-linked glucose backbone [ 9]. Despite the structural variability found in the species, the functions of the XyG in plants growth and development are hypothesized to be conserved among all species of flowering plants [ 10].
In dicotyledonous plants except for graminaceous, the cellulose and xyloglucan are in equal proportions. Some XyG chains are linked to the cellulose microfibrils supporting the important role of rigidity and maintenance of the cell, the rest XyG chains are cross-linked to cellulose microfibrils and pectic polymers, and altogether integrate the complex cell wall matrix. In addition XyG is thought to control cell wall enlargement potentially through the action of α-expansin, XyG endotransglucosylase or β-(1,4)-endoglucanases [ 11]. Several authors have revealed the XyG function by means of XyG-deficient mutants of
Xylans are a diverse group of polysaccharides with the common backbone of β-(1,4)- linked xylose residues, with side chains of α-(1,2) linked glucuronic acid and 4-
3.2.3. Mannans and glucomannans
The β-(1,4)-linked polysaccharides rich in mannose or with mannose and glucose in a non-repeating pattern are the glucomannans and galactoglucomannans. Even though their presence in primary cell wall is low, mannans have been studied in their role as seed storage compounds, as evidenced by the embryo lethal phenotype in an
Pectins represent an outstanding family of cell wall polysaccharides with extraordinary versatile, but not yet fully known structures and functions. In plants the functions of pectins fulfills important biological functions such as: growth, development, morphogenesis, defense, cell–cell adhesion, wall structure, signaling, cell expansion, wall porosity, binding of ions, growth regulators and enzymes modulation, pollen tube growth, seed hydration, leaf abscission, and fruit development [ 16]. The extracted pectins of citrus peel and apples are used as a gelling and stabilizing agent in food and cosmetic industries. Pectins within the fruits and vegetables are part of the daily dietary fiber and have multiple positive effects on human health including lowering cholesterol, serum glucose levels, decrease occurrence of diabetes and cancer [ 17- 19]. This points the relevance of pectins in diverse emerging fields of study, even in human health.
Pectins are the most wide complex family of polysaccharides in nature. They are present in primary walls of dicots and non-graminaceous monocots with approximately 35%; in grass and other commelinoid primary walls 2-10% and up to 5% in walls of woody tissues [ 20]. Pectins are formed with α-(1,4)-D-galacturonic acid residues. Galacturonic acid (GalA) comprises approximately 70% of pectin linked at the
Homogalacturonan (HG) is the most abundant polymer of the pectins, it comprises nearly the 60% of pectins in plant cell wall [ 20]. HG is formed by long chains of linear 1,4-linked α-D-galacturonic acid, some of the carboxyl groups are partially methyl-esterified at C-6 and acetyl-esterified at positions
3.3.2. Pectic branched polymers: Rhamnogalacturonans-I and Rhamnogalacturonans-II
Rhamnogalacturonan-I (RG-I) is a family of pectic polysaccharides that represent 20-35% of pectin. It contain a backbone of the repeating disaccharide galacturonic acid and rhamnose: [α-(1,2)-D-GalA-α-(1,4)-L-Rha] n partially substituted
Rhamnogalacturonans-II are the most complex and branched polysaccharides of pectin. RG-II is a minor pectic component of plant cell walls with between 0.5 to 8% in dicots, non-graminaceous, monocots, and gymnosperms, and less than 0.1% in primary walls of commelinoid monocots [ 26]. The RG-II has a characteristic structure of seven to nine residues of α-D-galacturonic acid backbone with four branches clearly differentiated designated A, B, C and D ( Figure 5).
RG-II has several kinds of substituents including 11 to 12 different glycosyl residues, some of them rare sugars in nature, like 2-
4. Biosynthesis of the plant cell wall polymers
Cell wall biosynthesis begins during cell division in the cytokinesis phase through the formation of the cell plate in the middle of the cell. Eventually, the primary cell wall is assembled by the deposition of polymers of cellulose, hemicelluloses and pectin. The biosynthesis of wall polymers starts in the nucleus with the transcription of genes coding for wall-related proteins and enzymes. Individual elements are channeled into the endomembrane system of endoplasmic reticulum and Golgi apparatus where they are polymerized and modified. The former polymers are then transported through vesicles and secreted outside plasma membrane for subsequent assembling and linkage to the wall. This mechanism is highly regulated along the process and depends on the physiological state of the cell and the interplay of signals going in and out of the cell [ 34].
Specifically, the long and rigid cellulose microfibrils of plant walls are synthesized from the inner face of the plasma membrane by cellulose synthase (CESA) complexes, which comprise multiple subunits forming a rosette structure of six globular CESA-containing complexes each of which synthesizes growing cellulose chains of 6–10 cellulose molecules (For review, see [ 4]). Newly synthesized microfibril is propelled by the action of the CESA, which polymerize the glucan chains in the specific positions driven by cortical microtubules [ 35]. Afterwards, microfibril is linked with xyloglucans (XyG) and pectic polysaccharides to form the cell wall complex network. Matrix polysaccharides not only cross-link microfibrils but also prevent the self association of new microfibrils into larger aggregates. XyG interacts with the formed microfibrils in the surface and also may be trapped inside them [ 36]. It has been observed that in primary walls, microfibrils linked to xyloglucan are smaller in diameter (less chain per fiber) than those in secondary walls. Besides, the binding between XyG and cellulose is known to weaken cellulose networks but increase their expansibility. The XyG is bound differently to three cellulose microfibrils domains. The first is available to endoglucanases, the second has to be solubilized by concentrated alkali and a third XyG is neither enzyme accessible nor chemical [ 36]. According to this, the type of hydrolysis used to obtain the fractions of polysaccharides (oligosaccharides) in some assays results in products of different degree of polymerization, which is related to some specific biological functions in the cell.
In contrast to cellulose, pectic polysaccharides are synthesized in the Golgi apparatus of the plant cell, and then are secreted to the apoplastic space through vesicular compartment. Polysaccharides are transported from cis-face to trans-face of the Golgi where they are sorted and packaged into vesicles of the trans-Golgi network for transport to the plasma membrane. The movement of the vesicles containing the polymers is presumably along actin filaments that have myosin motors. It is no clear, how the synthesis of the pectin polysaccharides is initiated or whether lipid or protein donors are involved. The possible modification of the pectic glycosyl residues may be esterification,
To complete the biosynthesis of polysaccharides, it is necessary the assembly of the transported elements to form the functional matrix. This event involves both enzymatic and non-enzymatic mechanisms in the apoplast [ 37]. The physicochemical properties existing in the wall are dependent on the hydrophobic and hydrophilic domains given by the water and solutes. The hydrophobic domain is formed by the link of cellulose microfibrils to the hydrogen bonds that lead the exclusion of water from the interacting chains. The hydrophobic interactions may also be controlled by enzymes that diminish the branching of the xyloglucan linked to cellulose microfibrils such as xylosidases and glucanases [ 38]. Meanwhile, the hydrophilic domain of the wall is given by pectin polymers. Together, both domains contribute to the protoplast matrix medium leading the rearrangement of some polymers as the homogalacturonan. Linear homogalacturonans are synthesized in a highly methyl-esterified form in the Golgi and transported to the wall in membrane vesicles to be desesterified by wall localized pectin methylesterases. The conversion of the HG from the methylesterified form to the negatively charged form has been associated with the decrease of growth [ 39].
The glycosiltransferases and hydrolases are enzymes localized in the Golgi apparatus and work together to produce the xyloglucan precursors. Some changes take place after the synthesis of hemicelulloses in the Golgi. It has been shown that a specific apoplastic glycosidases are responsible for the trimming of new xyloglycan chains and this determines the heterogeneity of the polymer in the cell wall [ 6]. Hydrolases are likely to play an important role in determining hemicelluloses structures in the cell wall, and are coexpressed with polysaccharide biosynthetic enzymes. For detailed information about the cell wall related enzymes see [ 4, 6, 10, 16, 40].
In the last decades many biochemical approaches have enabled the identification and characterization of the structure of cell wall polymers and the enzymes involved in their biosynthesis. Beside classical molecular analyses, the development of
5. Biological activity of plant cell wall derivatives
Exhaustive studies on the structure and function of the plant cell wall have led to the discovery of biologically active molecules derived from its polymeric carbohydrate components. These molecules are found in nature and can be released by acid, basic or enzymatic hydrolysis of the primary cell wall polysaccharides. Due to the complex combination of carbohydrate polymers in the cell wall of plants, there are variations among the physicochemical structure of hydrolyzed fragments, which exerts striking differences on activity and specificity to regulate some physiological processes in plants. These plant oligosaccharides with regulatory properties were called oligosaccharins and have been extensively studied by the workgroup of Albersheim since the mid-70s (reviewed in [ 41]). Among the oligosaccharins derived from plants, the most active and therefore the most studied are those derived from pectins and hemicelluloses, whose main regulatory functions depend on the degree of polymerization, chemical composition and structure, and can be divided in two broad categories: activation of plant defense mechanisms and plant growth and development.
In order to exert their regulatory properties, oigosaccharins must be first recognized by specific plant cell receptors which may be lectin-type proteins capable of transmitting the signal into the cell [ 42]. Even when the complete recognition mechanisms and signaling pathway for plant-derived oligosaccharins is far from being fully understood, protein receptors that recognize these molecules have been characterized in the model plant
5.1. Pectin-derived oligosaccharins
Even when the most abundant component of pectins is the galacturonic acid, partial depolymerization of the pectic polysaccharides generates fragments that may (or not) contain other residues such as rhamnose, galactose, arabinose, xylose, glucose and mannose [ 20]. This combination confers variability to the structure, and thereby to the biological activity of the oligosaccharins. The oligosaccharins derived from homogalacturonan are called oligogalacturonides (OGAs), which are linear oligomers of galacturonic acid, where some residues may be methyl-esterified or acetylated. OGAs are elicitors of defense responses in plants, triggering the synthesis and accumulation of phytoalexins (antimicrobial compounds) and other molecular indicators of the activation of defensive patterns, such as the induction of pathogenesis related proteins and genes related to the hypersensitive reaction [ 47]. OGA-induced defense response patterns are summarized in Table 1.
OGAs trigger the rapid accumulation of reactive oxygen species (ROS) in plants, which is necessary for the deposition of callose, polysaccharide produced in response to wounding and pathogen infection. Furthermore, ROS are signaling molecules of several intracellular events. Therefore it was proposed ROS were involved in the OGA-induced resistance against fungal pathogens in three different ways: (1) directly exerting a cytotoxic effect to the invading pathogen, (2) inducing callose deposition for reinforcing the plant cell wall, and (3) mediating the signals leading to the expression of defense related genes and defensive metabolites [ 48]. Nevertheless, recent findings in
|Induction of phenylalanine ammonia-lyase||9|||
|Induction of chalcone synthase||9-15|||
|Induction of -(1,3)-glucanase||3|||
|Protease inhibitors synthesis||2-3|||
|Induction of ethylene production||5-19|||
|Steem growth inhibition||8|||
|Protease inhibitors synthesis||10-14||[88,89]|
|Increase of the color and anthocyanin content||3-20|||
Plants treated with OGAs exhibit an enhanced resistance to pathogen infections. The induction of the defensive genes, peroxidase and β-(1,3)-glucanase has been related to the enhanced resistance of OGA-treated
The degree of acetylation and methylation of OGAs has been less addressed but emerging research showed the influence of these functional group substituents on plant defense responses. The effect of the degree of acetylation of OGAs on the elicitation of defenses in wheat (
Table 1 shows that OGAs modulate diverse growth and developmental processes in plants. Early responses related to the signaling transduction pathways comprise membrane depolarization, cytosolic acidification, apoplast alkalinization and calcium mobilization at the plasma membrane level, due to the activity of Ca 2+ channels. Calcium ions are very important second messengers in plants and its level in intracellular compartments is determinant for the kind of physiological response. In tobacco cells OGAs induced different patterns of Ca 2+ influx into cytosol, mitochondria and chloroplasts [ 54]. The increase in cytosolic free Ca 2+ has been proposed to mediate the regulation of stomatal aperture and production of hydrogen peroxide in the guard cells of tomato (
OGAs regulate morphogenesis in plant tissues in a process associated with the action of auxins, which are growth-regulating phytohormones. Particularly, OGAs and auxins appear to play an antagonist role; since OGAs inhibit the expression of some auxin-inducible genes steps downstream of the auxin perception [ 58]. In this sense, root differentiation induced by OGAs was studied in
Interestingly, the structure and stimulating activity of a rhamnogalacturonan I-derived oligosaccharide (RG-IO) isolated from flowers of
5.2. Hemicellulose-derived oligosaccharins
Xyloglucan is the main hemicellulosic component of the plant cell wall. Biological effects of xyloglucan derivatives are related to the intrinsic physiological function of polymeric xyloglucan in plant cells, comprising the control of extensibility and mechanics of the cell wall and cell expansion. Most research in this field highlights their regulatory activity on cell growth and elongation, which relies in the molecular size, distribution, and levels of substituted xylosyl units with galactosyl and fucosyl residues [ 63]. It has been observed active xyloglucan oligomers (XGOs) accelerate cell elongation in peeled stem segments of
Xyloglucan-derived octasaccharides promoted growth of coleoptiles in wheat seedlings and induced a rapid increase of α-L-fucosidase activity in
On the other hand, galactoglucomannan is composed by a backbone of glucose and mannose residues with side chains of galactose. More recent research about cell wall oligosaccharides derivatives has demonstrated a growth-regulating activity of galactoglucomannan-derived oligosaccharins at very low concentrations. Galactoglucomannan oligosaccharides (GGMOs) modulate root morphology in mung bean (
Furthermore, GGMOs inhibited the elongation induced by exogenous phytohormones of pea stem segments [ 74], root and hypocotyl growth of mung bean [ 71, 73] and
5.3. Cellulose-derived oligosaccharins
During many years it was thought that only non-cellulosic oligosaccharides derived from plant cell wall were biologically active. Surprisingly, it has been recently demonstrated that fragments of oligosaccharides released during cellulose degradation, called cellodextrins (CD), induce a variety of defense responses in grapevine cells. CD are oligomers of linear β-(1,4)-linked glucose residues. The induction of oxidative burst, transient elevation of cytosolic Ca 2+, expression of defense-related genes, and stimulation of chitinase and β-1,3-glucanase activities were triggered by CD in grapevine cells. Also, CD oligomers with a degree of polymerization ≥7 enhanced protection in detached leaves of grapevine against
6. Current applications of plant cell wall-derived oligosaccharins
Increasing knowledge of the factors that modulate the biological activity of cell wall-derived oligosaccharides has naturally led to the development of technologies aimed to exploit the potential of these molecules in different fields. The first successful applications occurred in agriculture, where different crops can now be treated with commercially available preparations of pectin-derived-oligosaccharins in order to enhance the basal resistance of plants, and decrease the possible losses related to phytopatogenic infections. Another alternative is the use of oligosaccharins to improve the yield, as seen in tomato (
The authors appreciate the support of Juan Reyes Calderón with the edition of the figures shown in this chapter.