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Tesi di LAUREA SCIENZE AGRARIE - Metabolic analysis of Arabidopsis plants with altered lignification

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Tesi di LAUREA SCIENZE AGRARIE - Metabolic analysis of Arabidopsis plants with altered lignification


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Tesi di LAUREA

Metabolic analysis of Arabidopsis plants with altered lignification


Lignin is, after cellulose, the second most abundant terrestrial biopolymer, accounting for approximately 30% of the organic carbon in the biosphere. The ability to synthesize lignin has been essential in  the  evolutionary adaptation of  plants from an aquatic environment to land. Lignin is crucial for structural integrity of the cell wall and stiffness and strength of the stem. In addition, lignin waterproofs the cell wall, enabling transport of water and solutes through the vascular system, and plays a role in protecting plants against pathogens.

Lignin is an aromatic heteropolymer, abundantly present in the walls of secondary thickened cells.

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic- pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H2O2, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The “randomness” of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may  derive  in  part  from  monomers  and  conjugates  other  than  the  three  primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate.

We have profiled the methanol-soluble oligolignol fraction of Arabidopsis thaliana stem, a tissue with extensive lignification. Two genes have been modified to study the variation in lignin composition, F5H and COMT, respectively up and downregulated.

At the beginning, we deeply worked on a new liquid chromatography machine (UPLCTM) optimizing the several parameters which permit an optimal separation of the molecules.

This operation took us a lot of time but these technical parameters are now used by the research group. Using liquid chromatography-mass spectrometry, we tried to discover new monomers being generated from the reshape monomer pathway. Although we could not present any new molecule, we had experience about machine settings and laboratory pratical skills.

To describe the practical aspects of my work, in this thesis I am presenting a research on poplar lignins made by the same research group I worked with.


The cell represents the morphologic and functional unit of all the living organisms. Eukaryotic cell presents a highly compartimental cellular organization: this is due to the enormous membranes systems development which is highly specialized. Cell nucleus, mitochondrions, chloroplasts, endoplasmic reticulum and Golgi apparatus are delimitated by membranes. The cellular portion excluded from these compartment is called cytoplasm.

Plant cells are quite different from the cells of the other eukaryotic kingdoms' organisms. Their distinctive features include:

           a  large  central  vacuole,  which  maintains  the  cell's  turgor  and  controls movement of molecules between the cytosol and sap, which stores useful material and waste;

            cell wall composed of cellulose, and in many cases lignin, and deposited by the protoplast on the outside of the cell membrane;

            chloroplasts, organelles that absorb light and use it in conjunction with water and carbon dioxide to produce sugars, the raw material for energy and biomass production in all green plants.

Figure 1

Plant cell. Major components are highlighted with the arrow. Cell wall is the external part of the cell (green coloured).

Plasma membrane

The plasma membrane is a semipermeable lipid bilayer found in all cells, that surrounds the cytoplasm. The barrier is selectively permeable and able to regulate what enters and exits the cell, thus facilitating the transport of materials needed for survival.

Figure 2

Plasma membrane. Note the lipid bilayer constituted of hydrophilic region (yellow) and hydrophobic tales



Chloroplasts are organelles found in plant cells that conduct photosynthesis. Chloroplasts absorb light and use it in conjunction with water and carbon dioxide to produce sugars, the raw material for energy and biomass production in all green plants (see figure below).

Endoplasmic reticulum

The endoplasmic reticulum is an organelle found in all eukaryotic cells that is an interconnected network of tubules, vesicles and cisternae. These structures are responsible for several specialized functions: protein translation, folding, and transport of proteins to be used in the cell membrane, or to be secreted from the cell, production and storage of glycogen, steroids, and other macromolecules. The general structure of the endoplasmic reticulum is an extensive membrane network of cisternae. The two varieties are called rough endoplasmic reticulum (rER) and smooth endoplasmic reticulum (sER). The surface of  the  rough endoplasmic reticulum is  studded with  protein-manufacturing ribosomes giving it a 'rough' appearance. The rough endoplasmic reticulum works in concert with the Golgi complex to target new proteins to their proper destinations. The rER is key in producing:

           lysosomal enzymes with a Mannose-6-phosphate marker added in the cis-Golgi network

           secreted  proteins,  either  secreted  constitutively  with  no  tag,  or regulated secretion involving clathrin and paired basic amino acids in the signal peptide.

           integral membrane proteins that stay imbedded in the membrane as vesicles exit and bind to new membranes.

Golgi apparatus

The Golgi apparatus is an organelle found in most eukaryotic cells. The primary function of the Golgi apparatus is to process and package the macromolecules such as proteins and lipids that are synthesized by the cell. It is particularly important in the processing  of   proteins  for   secretion.  The   Golgi  apparatus  forms   a   part   of   the endomembrane system of eukaryotic cells. The Golgi is composed of membrane-bound sacs known as cisternae. Vesicles from the endoplasmic reticulum (via the vesicular- tubular cluster) fuse with the cis-Golgi network and subsequently progress through the stack to the trans-Golgi network, where they are packaged and sent to the required destination. Each region contains different enzymes which selectively modify the contents depending on where they are destined to reside.

Figure 3

Endoplasmic reticulum and Golgi apparatus. Proteins, synthetized by the ribosomes on the endoplasmic

reticulum, pass through the cisternae of the Golgi apparatus to be prepared to be secreted.


In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. Their origin is unclear, but according to the endosymbiotic theory, mitochondria are thought to be descended from ancient bacteria. These organelles range from 1–10 micrometers (µm) in size. Mitochondria are sometimes described as 'cellular power plants' because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. The organelle is comprised of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix.

Figure 4

Structure of a mitochondrion. Note the huge development of membranes which are immersed in the matrix.

1.2. Cell Wall

Cell wall is a complex polymeric structure characteristic of plant cell. It is a fairly rigid layer surrounding a cell, located external to the plasma membrane, which provides the cell with structural support, protection, and acts as a filtering mechanism. Its chemical composition, tridimensional organization and properties can vary with growth and development of the cell.

The plant cell wall has three components:

               the middle lamella, a layer rich in pectins. This is the outermost layer, forming the interface between adjacent plant cells.

               the primary cell wall, a wall formed during cell division that expands as the cell grows. It keeps the cell from taking up too much water.

               the secondary cell wall, a thick layer formed inside the primary cell wall after the cell is fully grown. It is not found in all cell types.

Cells of the same tissue have a characteristic cell wall and often cells functions are determined by the cell wall. Since the cell wall is external to the plasma membrane, synthesis, transport and secretion of its components plan complex mechanism linked to the cell metabolism and functionality of the endomembranes (endoplasmic reticulum, Golgi apparatus, plasma membrane and vesicles).

Cell wall is a dynamic structure fully involved in plants development and life with a lot of functions:

            determines cells shape, dimension and growth rate

            resists to the turgor pressure of the protoplast

            confers rigidity to the cell performing in the plant the same role of the skeleton in the animals

            acts as a physical and biological barrier against pathogens

            represents the most important renewable biomass on the earth

            represents the most important fiber source for humans diet.

Between the cell wall and the protoplast, there is a continuous information interchange which permit to vary cell walls chemical-physical properties in response to enviromental conditions, growth, cell development.

Cell wall has a various permeabilty degree. In the pores, whose dimensions vary between 3.5-5 nm., can diffuse water and low molecular weight molecules such as: ions, sugars, aminoacids, hormons.

In the entire plant is set a continuous flow of water and solutes passing by the pores, in a place called apoplast. The apoplastic flow is considerably limitated by the cell walls intrusion by hydrophobic polymers such as lignin, suberine, cutine.

Figura 5

Detail of the plant cell. Cells can communicate with pores called plasmodesmata.

1.2.1. Genesis and development

Cell walls genesis in plants starts during cell division but its growth and development continues, following a precise genetic planning, until the cell stops being active. Cell wall starts forming when a large number of microfilaments arrange in a characteristic barrel shape in the equatorial plane of the spindle apparatus. This structure is characteristic of plant cell and it is called phragmoplast. It is constituted of two opposite groups of microfilaments, whose fast growth ends are organized in a central position of the division plate, while the slow growth ends are orientated to the opposite direction.

Next to the phragmoplast are present a large number of endoplasmic reticulum and dictisomes elements; these structures produce a great number of vesicles which contain the polymeric material used for cell walls building.

Cell plate is organized by the action of phragmoplasts microfilaments, and it derives from the progressive fusion of the vesicles containing pectin.

Plasma membrane of the daughter cells is formed from the vesicles’ fusion while their contents form the cell plate. In this way, cell plate grows to reach the wall of the mother cell, the new formed plasma membranes fuse with the mother cells one and two new cells are now separeted.

Cell  plate  is  made  of  three  layers:  the  central  portion  constituted  of  pectin, represents the middle lamella. It derives from the assembly of the polymeric material transported by the vesicles produced by Golgi apparatus. In the external layers are present cellulose microfibrils; they are synthetized by a multienzymatic complex (cellulose synthase) which is present in the neo-formed plasma membranes. This event is the starting point of the primary walls biogenesis which is directed by the two daughter cells. Their protoplasts can communicate by cytoplasmatic channels highly organized called plasmodesmata. When the cell plate is complete, primary walls growth and development continues by the synthesis of pectins, hemicelluloses, proteins which constitute the matrix, and cellulose which constitute the fibrillar material.

Matrix polysaccharidesare synthetized by Golgi apparatus and endoplasmic reticulum, concentrated in vesicles and released in the primary wall.

Cellulose’s microfibrils, synthetized by multienzymatic complex which are integrated in the plasma membrane, are directly secreted in the primary wall.

Depending on the role in the plant, cells, once they ultimate their development, present a cell wall differently organized. Some cells, such as parenchyma cells, are only constituted of primary wall while highly specialized cells, such as mechanic elements (fibers, sclereids) and vessel elements, present the secondary wall essential for their functions.

1.2.2. Middle lamella

Middle lamella links 2 adjacent cell walls: its partial hydrolysis determines the intercellular space’s formation. Middle lamella’s thickness is about 0.1 µm.: it is composed by pectines and water; some enzymatic proteins could be present in this structure.

Pectins are complex polysaccharides constituted by acid polymers: rhamnogalacturonan I and II, homogalacturonan and neutral polymers. Rhamnogalacturonan I is the major constituent of the pectins. The chemical structure of this polymers is made of galacturonic acid and rhamnose residuals’ chain. Side neutral chains of arabinose and galactose are linked to rhamose’s residuals which have a key role to keep the water linked.

Homogalacturonan are constituted almost completely of   galacturonic acids residuals linked with α 1 4 bonds. The function of this polymers seems not to be only structural, homogalacturonan can have a regulatory role in the tissues.

Pectins are able to form reversible gel and pasty solutions with water, this property is linked to their role in the cell wall. The quantity of water linked depends on the quantity of pectins: hydratation rate influences cell walls mechanical properties.

1.2.3.  Primary wall

Primary  wall  is  a  highly  organized  structure,  characterized  by  a  continuous interaction between a high resistence fibrillar material and an extended matrix.

The fibrillar material of plants primary wall, is represented by cellulose: this polymer  represents  the  most  abundant  polysaccharide  on  earth.  Matrix  is  composed

essentially of polysaccharides such as pectins and hemicellulose, enzymatic and structural proteins, water and a low percentage of lipids. Matrixs composition may vary between 2 different species but even from 2 different tissues.

Cellulose is the fibrillar component of the cell wall. It derives from the association of long glucanic chains constituted of glucosidic residuals linked with β 1→ 4 bonds.

β 1→ 4 bonds formation requires a 180° rotation of a glucose molecule in respect to the other one; as a consequence the constitutive unit of the glucanic chain is represented by the disaccharide cellobiose 1→ 4 glucosidic bond confers to the glucanic chain a structure similar to a flattened tape.Chain is stabilized by hydrogen bonds organizing between the glucosidic units.

Glucose units of different chains may organize hydrogen bonds, in this way a large number of glucanic chains aggregate together and form unsoluble fibrils called cellulose microfibrils. The union of several microfibrils form cellulose macrofibrils.

Primary walls pectins are constituted by the same polymers which compose the middle lamella.

Hemicellulose is a heterogeneous group of polysaccharides which interact with cellulose microfibrils and the other matrixs polymers.

There are several kind of hemicellulose and their composition vary between species and during cell development.

Xyloglucans represent the major constituent of hemicellulose and they are constituted of a linear chain of glucose with  β 1 4 bonds; 6th carbon of  almost all the glucoses residuals are linked to xylose residual with α 1→ 6 bonds.

Xylans are constituted of a linear chain formed by 2 xylose units with β 1→ 4 bonds.L-arabinose and galacturonic acid may often band to xylose originating polysaccharide.

Xylans can link to cellulose by means of hydrogen bonds among xylanic chains’ xylose and glucanic chains’ glucose. Lateral residuals’ presence prevent this bonds. So, the more the residual presence is high, the lower the xylans will aggregate to cellulose.

Figura 6

Organization of the cell wall.Cell is divided from the outside by the plasma membrane (lower layer).

A complex reticulum of pectins, hemicelluloses and cellulose costitute the cell wall.

1.2.4. The importance of water in the cell wall

Water is a fundamental component of the primary cell wall in which it represents about 60% of the fresh weight. It is involved in several functions and its presence in the cell wall has a big influence in properties as chemical and physical polymeric components’ as well as in the mechanical charateristic of the cell wall. Water has a structural function because it partecipates at the pectin polysaccharide gel formation. Glucanic chains which constitute cellulose microfibrils are kept together by hydrogen bonds. Matrixs hemicellulose and cellulose may be keep together by hydrogen bonds.

Water presence may reduce the bond power between cellulose microfibrils and hemicelluloses influencing cell walls mechanical properties.

Water determines cell walls permeability rate and it allows ions presence which can form salts with acid polysaccharides. It has a role in stabilizing the secondary structure of the matrixs   macromolecules and it represents the mean in which all the enzymatic reactions may take place in the cell wall. Water is a fundamental factor for the cell wall plasticity which depends on the hydration rate of the cell wall itself.

Once the cell wall has finished its development, in some tissues, it may become an extremely rigid structure because matrixs water is completely replaced by lignin, a hydrophobic polymer.

1.2.5. Secondary wall

In some tissues, cell walls final development presents only  the primary cell wall. In some other tissues such as fibers and sclereids of the mechanical tissue, xylematic vases and stomata’s guard cells present successive layers of polysaccharidic material on the primary wall which constitute the secondary wall.

Secondary wall  is  composed  of  a  scarce  matrix  interacting with  an  abundant fibrillar material made of cellulose. In secondary wall there are no pectins or proteins while hemilcelluloses are abundant but chemically different from primary cell wall ones.

Water level is always very low, as a consequence of the scarce presence of matrix and the low presence of pectins. Cellulose is the main element of the secondary cell wall, normally 60-70% of the dry weight. Cellulose microfibrils are disposed parallel to each other: this kind of disposition is called parallel weave.

Cellulose  microfibrils  present  a  higher  polymerization  level  compared  to  the primary cell walls ones.Their diameter is always higher in the secondary wall.

Matrix is mostly composed of hemicelluloses. Angiosperm secondary walls hemicellulose is very different from gymnsperm one. In gymnosperms hemicellulose are predominant glucomannans ad galactoglucomannans. These polysaccharides present a linear structure formed of mannose and glucose units linked with β 1→ 4 bonds, the ratio mannose:glucose is 3:1.

For what concern angiosperms secondary wall, it is mostly constituted of 4-O- methyl-glucuronoxylans.These polysaccharides are made of linear chains formed of two xylose units linked with β 1→ 4 bonds.

Secondary wall is constituted of three layers called s1, s2, s3. This characteristic organization is caused by the cellulose fibrils’ orientation.

S1 layers thickness vary between 0.1 and 0.65 µm., depending on the tissue type: tracheid, fiber,     summer or vernal vessel element. It is the first layer internally linked to the primary wall and it is composed of cellulose microfibrils’  lamellas associated to hemicelluloses.

Every lamella is organized with a helicoidal type parallel weave, microfibrils are orientated with a high angle compared to cell axis.

S2 follows s1, normally very thick (1,5-7 µm.), constituted of a high number of cellulose microfibrils’ lamellas associated to hemicelluloses. Even in this case, lamellas are organized with a helicoidal type parallel weave but the angle formed with the cell axis is very little.

S3 layer presents a thickness of  0.1 µm.; cellulose microfibrils and hemicelluloses’

orientation is the same as the s1 one.

Secondary wall is not equally disposed on the primary walls surface, it is internally organized following a genetic model. Secondary wall is disposed on vessel element following characteristic models.

Figure 7

Structural organization of the cell walls of fibers

1.2.5. Polysaccharides biosynthesis

Structural cell wall polysaccharides and stock polysaccharides are constituted of a variable number of sugars(monosaccharides) linked with glucosidic bonds.

All the sugars present hydroxide groups (-OH) and one aldehyde group (-CH=O) or ketone group (-C=O). When the hydroxide group of a sugar linked to carbon 1 reacts with one of the hydroxide group of another sugar, a glycosidic bond is formed with the elimination of a water molecule.

Depending on the hydroxide groups involved in the glycosidic bond formation, there are several kind of bond, consequently several disaccharides structurally different.

Successive addiction of sugars sythetizes oligosaccharides and polysaccharides. These    are    synthetized    by    enzyme    or    multienzymatic    complexes             called

polysaccharide synthase. These enzymes are glycosyltransferases which are integrated in the endomembranes’ system (endoplasmic reticulum, Golgi apparatus, plasma membrane).

Glycosidic residual are transferred from a diphosphate nucleoside sugars to the rising polysaccharide chain.

Lignin, cutine and suberine are three polymeric components which interact in different ways with the cell wall polysaccharides which constitute some specialized tissues. The presence of these polymers, highly modifies physical and chemical cell walls properties and cell functionality.


Lignin is, after cellulose, the second most abundant terrestrial biopolymer, accounting for approximately 30% of the organic carbon in the biosphere. The ability to synthesize lignin has been essential in  the  evolutionary adaptation of  plants from an aquatic environment to land.

Lignin fills the spaces in the cell wall between cellulose, hemicellulose and pectin components, especially in tracheids, sclereids and xylem. It is covalently linked to hemicellulose and  thereby crosslinks different  plant  polysaccharides, conferring mechanical strength to the cell wall and stiffness and strength of the stem (23, 71).

In addition, lignin waterproofs the cell wall, enabling transport of water and solutes through the vascular system, and plays a role in protecting plants against pathogens.

The polysaccharide components of plant cell walls are highly hydrophilic and thus permeable to water, whereas lignin is more hydrophobic. The crosslinking of polysaccharides by lignin is an obstacle for water absorption to the cell wall. Thus, lignin makes it possible for the plant's vascular tissue to conduct water efficiently.

Although researchers have studied lignin for more than a century, many aspects of its  biosynthesis  remain  unresolved.  The  monolignol  biosynthetic  pathway  has  been redrawn many times and remains a matter of debate (36, 67). Likewise, the biochemical processes leading to dehydrogenation of the monolignols in the cell wall and their polymerization and deposition are fields of active discussion (31, 60, 81, 122, 128). In addition, we are only beginning to understand the transcriptional and post-translational mechanisms and metabolic complexes regulating the flux through the phenylpropanoid and monolignol biosynthetic pathways. Lignin is present in all vascular plants, but not in

bryophytes, supporting the idea that the original function of lignin was restricted to water transport.

Lignin is first deposited in the middle lamella and the cell corners of the primary wall after the formation of the secondary wall has started, at the so-called nucleation sites, from which the lignin polymers can grow. The nature of these nucleation sites is unknown. Ferulates, conjugated to polysaccharides, and their dehydrodimers are well established as being incorporated into grass lignins. There is some evidence that ferulates and diferulates may act as attachment sites for monolignols (109). Structural cell wall proteins rich in aromatic residues, such as glycine-rich proteins (76), may have a similar function. It is interesting to note that several apoplastic peroxidases from zucchini and horseradish bind

pectin in their Ca2+- induced conformation (101); one such peroxidase has been cloned

(19,20). Given that the middle lamella and the cell corners are rich in Ca2+ pectate (21) and are the first sites to be lignified, Ca2+  pectate-bound peroxidases may conceivably play a role in the spatial control of lignin deposition, and changes in Ca2+ and H+ concentrations may modulate the location of these peroxidases. The negatively charged pectins are also good binding sites for polyamines (19) and, hence, may be suitable sites for H2O2 generation by polyamine oxidases (94). It is tempting to speculate that pectin-binding peroxidases and polyamine oxidases may act locally in the early stages of lignin deposition

both for H2O2 generation oxidation of monolignols, cinnamic acids bound to polysaccharides or polyamines, or aromatic residues on certain proteins, such as glycine- rich proteins (76).

2.1. Composition and structure

Lignins are complex racemic aromatic heteropolymers derived mainly from three hydroxycinnamyl  alcohol  monomers  differing  in  their  degree  of  methoxylation,  p- coumaryl M1H, coniferyl M1G, and sinapyl M1S alcohols (figure below).

These monolignols produce, respectively, p-hydroxyphenyl H, guaiacyl G, and syringyl S phenylpropanoid units when incorporated into the lignin polymer. The amount and composition of lignins vary among taxa, cell types, and individual cell wall layers and are influenced by developmental and environmental cues (18). Although exceptions exist, dicotyledonous angiosperm (hardwood) lignins consist principally of G and S units and

traces of H units, whereas gymnosperm (softwood) lignins are composed mostly of G units with low levels of H units.

Lignins from grasses (monocots) incorporate G and S units at comparable levels, and more H units than dicots (11). Lignification is the process by which units are linked together via radical coupling reactions (50, 126). The main “end-wise reaction couples a new monomer (usually a monolignol and usually at its β-position) to the growing polymer, giving rise to structures A, B, and D2 (all of which are β-linked).

Coupling between preformed lignin oligomers results in units linked 5–5 D and 5– O–4 E. The coupling of two monolignols is a minor event, with resinol (β–β) units C or cinnamyl alcohol end groups X1 as the outcome. Monolignol dimerization and lignin are substantially different processes (2), explaining why lignification produces frequencies of the various units that are different from those produced by dimerization (Figure 8). The interconnections are described in Figure 9. The most frequent inter-unit linkage is the β–O–

4 (β-aryl ether) linkage A. It is also the one most easily cleaved chemically, providing a basis for industrial processes, such as chemical pulping, and several analytical methods. The other linkages are β –5 B, β–β C, 5–5 D, 5–O–4 E, and β–1 F, which are all more resistant to chemical degradation. The relative abundance of the different linkages depends largely on the relative contribution of a particular monomer to the polymerization process. For example, lignins composed mainly of G units, such as conifer lignins, contain more resistant (β–5 B, 5–5 D, and 5–O–4 E) linkages than lignins incorporating S units because of the availability of the C5 position for coupling.

Figure 8

Lignification differs substantially from simple dimerization of monolignols.

(a) Dimerization of coniferyl alcohol produces only three dimers, in each of which at least one of the coniferyl alcohols is coupled at its β position. The 5–5 and 5–O4 dimers shown in most texts (and crossed out) do not actually arise in any significantway from monomer dimerization reactions. The new bond formed by the radical coupling reaction is noted in bold.

(b) Crosscoupling of coniferyl alcohol with a G unit gives only two main products, explaining why there are more β-ethers formed during lignification than in monolignol dimerization (or in synthetic dehydrogenation polymer (DHP) synthesis, where dimerization is too frequent).

Coupling of preformed oligomers is the source of most of the 5–5- and 5–O4 units. Sites of further coupling reactions during lignification are indicated by dotted arrows. Not shown: Sinapyl alcohol analogously dimerizes to only two products (not the β5 analog), and polymerization between a monolignol (either coniferyl or sinapyl alcohol) and an S unit in the polymer has only one outcomethe βO4 unit, explaining why high S lignins have elevated β-ether levels. Neither 5–5 nor 5–O4 units can be formed between S units; cross-coupling of G and S units can furnish 5–O–4-linked structures.

It is becoming clear that lignins are derived from several more monomers than just the three monolignols M1 (bold in Figure 9). Many normal plants contain lignins substantially derived from other monomers, and all lignins contain traces of units from apparently incomplete  monolignol  biosynthesis  and  other  (side-)  reactions  that  occur during that biosynthesis (113). Many of these units have been recently identified by their more substantial incorporation into lignins in transgenic and mutant plants with perturbations in the monolignol biosynthetic pathway.

Variously acylated lignin units Y were suspected to derive from acylated monolignols M9–M11 (95). p-coumarates Y3 on grass lignins are regio-specifically attached to the γ position of lignin sidechains, and on all types of lignin units, suggesting that they are not products of postlignification derivatization (85). Recent identification of novel ββ-coupling products from kenaf lignins that could have only arisen from sinapyl

acetate M9S (i.e., preacetylated sinapyl alcohol) provides more compelling evidence (86) and suggests that all of the acylated lignins (p-hydroxybenzoates Y2 in poplars, palms, and willows; p-coumarates Y3 in all grasses; and acetates Y1 in palms and kenaf, as well as the low levels in many hardwoods) derive from acylated monolignols M9–M11. Because acetylated components can represent a significant part of the polymer (over 50% of kenaf bast  fiber lignin units  are  acetylated, for  example), they should be  considered to  be authentic lignin monomers. Ferulates M4G, and their dehydrodimers that derive from radical coupling reactions and generate polysaccharide-polysaccharide cross-linking, are also intimately incorporated into lignins, particularly in grasses where they appear to function as nucleation sites for lignin polymerization (69, 109). Tyramine ferulate M8G (and possibly other hydroxycinnamate analogs) is intimately polymerized into the polymer in normal tobacco and is particularly enhanced in cinnamoyl coenzymeA (CoA) reductase (CCR)-deficient transgenic tobacco (110).

Dihydroconiferyl alcohol (DHCA) X5G and derived guaiacylpropane-1,3-diol units X6G are always detectable in gymnosperm lignins, suggesting that the monomer M5G is always produced with coniferyl alcohol M1G (111). DHCA-derived units X5G and X6G are major components of the lignin in a cinnamyl alcohol dehydrogenase (CAD)-deficient pine mutant where about half are involved in 55-coupled structures D (114). Similarly, cinnamyl X2 and benzyl X3 aldehyde groups are always detected in lignins. Whether they arise from postlignification oxidation reactions or from incorporation of the aldehyde monomers into the polymer is unclear, but the latter is highly implicated by the recent observations of increased levels in CAD-deficient plants, which also display products of endwise hydroxycinnamyl aldehyde incorporation into the growing polymerthe hydroxycinnamyl aldehyde 8–O–4-linked units K, particularly predominant in CAD- deficient angiosperms (76a, 113). Incorporation profiles of   hydroxycinnamyl aldehydes M2 provide further evidence that lignification reactions are under simple chemical control. In tobacco, sinapyl aldehyde M2S is found 8–O4-coupled (in structures K) to both G and S units, whereas coniferyl aldehyde M2G is only found cross-coupled to S units. Coniferyl aldehyde has not been successfully coupled to guaiacyl models in vitro, although the 8–O–

4-coupled dehydrodimer can be obtained.

An important corollary is that hydroxycinnamyl aldehydes are incorporated intimately into angiosperm GS lignins but only poorly into gymnosperm G lignin. The

most striking example of lignins incorporating substantial quantities of a monomer derived from truncated monolignol biosynthesis is in caffeic acid-O-methyltransferase (COMT)- deficient angiosperms (113). Plants severely depleted in COMT produce little sinapyl alcohol M1S but essentially substitute it with a monomer derived from its unmethylated precursor, 5-hydroxyconiferyl alcohol M15H. The incorporation is typically end–wise, but the o-diphenol results in novel cyclic structures, benzodioxanes J, in the lignin (112). As with other products, such units can be found at very low levels in normal plants.

Figure 9

Lignin monomers and structures in the polymer. (Monomers) Lignins derive primarily from the three traditional monolignols, the hydroxycinnamyl alcohols: M1H, M1G, and M1S. The fourth hydroxycinnamyl alcohol, M15H, is a significant monomer in COMT-deficient plants. Aldehydes M2 and M3 probably incorporate into all lignins and increasingly so in CAD-deficient plants. Hydroxycinnamate esters M4, particularly ferulates M4G, incorporate into lignins in grasses. Dihydroconiferyl alcohol M5G, and the guaiacylpropane-1,3-diol M6G derived from it, are monomers in softwood lignification and are highly elevated in a CAD-deficient Pinus taeda mutant; whether the syringyl analogs are also produced and incorporated into angiosperms is not known. Arylglycerols M7 are implicated by glycerol structures X7 in lignins, but may come from the isolation process. Variously acylated monolignols M9–M11 are implicated in many plants. Tyramine hydroxycinnamate M8, particularly tyramine ferulate M8G, appears to be a monomer in tobacco lignins. Note that it is conventional to use α, β, and γ for the side chain positions in the hydroxycinnamyl alcohols M1 and related products, but 7, 8, and 9 for the analogous positions in hydroxycinnamyl aldehydes M2 and hydroxycinnamate esters M4 and M8. Bracketed compounds have not been established as authentic monomers. (Lignin Polymer Units) Units are generally denoted based on the methoxyl substitution on the aromatic ring as H, G, S (and 5H); dashed bonds represent other potential attachments via coupling reactions. The most common structures in lignins from normal and transgenic plants are shown as structures AL with the bond formed during the radical coupling step (in bold); p- hydroxyphenyl

units are not shown. The dashed bonds indicate substitutions by methoxyl (in syringyl components) or other attachments from coupling reactions; generic side chains are truncated (zigzag lines). Most units arise from cross-coupling reactions of a monomer with the growing polymer or by polymer-polymer coupling reactions. Resinol units C are from monolignol-monolignol coupling (followed by further cross-coupling reactions). Most 5–5-linked units D are in the form of dibenzodioxocins D2. bis-Aryl ether units A2 are rare in most lignins, but relatively prevalent in tobacco. Units A3 are seen in isolated lignins but may result from the isolation process. Units F, β-1 structures, may not occur in lignins as drawn but as spirodienones, for example (129). Benzodioxanes J are the main units resulting from the incorporation of 5-hydroxyconiferyl alcohol M15H monomers into lignins, particularly in COMT-deficient angiosperms.Units K are prevalent in CAD- deficient angiosperms and arise from endwise coupling of hydroxycinnamyl aldehydes M2 into lignins. Unit L is a single example of a ferulate-monolignol cross-coupling product seen in grass lignins. (End Groups) End groups arise from coupling reactions that are not at the sidechain β -position. Hydroxycinnamyl end groups X1 arise from dimerization reactions and represent only a small percentage of the units (2). End groups X2 to X6 derive from the corresponding

monomers M2–M6; X6b is possibly an isolation artifact from oxidation of X6 units.

Glycerols X7 may be from monomers M7 or may be produced during ball milling from β-ether units A. - Acylated Units) Any of the units AL bearing a γ -OH may also bear an acyl group, partial structures Y1–Y3. They arise almost certainly from the corresponding monolignols M9–M11. (Miscellaneous) Finally, some other groups resulting from incorporation reactions are not accommodated by the other structures. Partial structures Z1 arise from incorporation of monomer M8; general aldehydes an arise from hydroxycinnamyl aldehyde monomers M2; general esters result from the incorporation of ferulates and dehydrodiferulates.

Lignin deposition is one of the final stages of xylem cell differentiation and mainly takes place during secondary thickening of the cell wall (37). Generally, secondary cell walls consist of three layers: the outer (S1), middle (S2), and inner (S3). Lignin deposition proceeds in different phases, each preceded by the deposition of carbohydrates, and starts at the cell corners in the region of the middle lamella and the primary wall when S1 formation has initiated. When the formation of the polysaccharide matrix of the S2 layer is completed,  lignification  proceeds  through  the  secondary  wall.  The  bulk  of  lignin <