Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Cell Walls - Structure & Function

I. Functions of the cell wall:   The cell wall serves a variety of purposes including:

1. maintaining/determining cell shape (analogous to an external skeleton for every cell).  Since protoplasts are invariably round, this is good evidence that the wall ultimately determines the shape of plant cells.
    
2. Support and mechanical strength (allows plants to get tall, hold out thin leaves to obtain light)
    
3. prevents the cell membrane from bursting in a hypotonic medium (i.e., resists water pressure)
    
4. controls the rate and direction of cell growth and regulates cell volume
    
5. ultimately responsible for the plant architectural design and controlling plant morphogenesis since the wall dictates that plants develop by cell addition (not cell migration)
    
6. has a metabolic role (i.e., some of the proteins in the wall are enzymes for transport, secretion)
    
7. physical barrier to: (a) pathogens; and (b) water in suberized cells.  However, remember that the wall is very porous and allows the free passage of small molecules, including proteins up to 60,000 MW. The pores are about 4 nm (Tepfert & Taylor 1987)
    
8. carbohydrate storage - the components of the wall can be reused in other metabolic processes (especially in seeds).  Thus, in one sense the wall serves as a storage repository for carbohydrates
    
9. signaling  - fragments of wall, called oligosaccharins, act as hormones. Oligosaccharins, which can result from normal development or pathogen attack, serve a variety of functions including:  (a) stimulate ethylene synthesis; (b) induce phytoalexin (defense chemicals produced in response to a fungal/bacterial infection) synthesis; (c) induce chitinase and other enzymes; (d) increase cytoplasmic calcium levels and (d) cause an "oxidative burst".  This burst produces hydrogen peroxide, superoxide and other active oxygen species that attack the pathogen directly or cause increased cross-links in the wall making the wall harder to penetrate.  
   
       Let's look at how this system works.  Consider a pathogenic fungus like Phytophthora.  In contact with the host plant the fungus releases enzymes such as pectinase that break down plant wall components into oligosaccharins.  The oligosaccharins stimulate the oxidative burst and phytoalexin synthesis, both which will deter the advance of the fungus.  In addition, the oligosaccharins stimulate chitinase and glucanase production in the plant.  These are released and begin to digest the fungal wall.  Fragments of fungal wall also act as oligosaccharins in the plant to further induce phytoalexin synthesis.  Cool!
    
10. recognition responses - for example: (a) the wall of roots of legumes is important in the nitrogen-fixing bacteria colonizing the root to form nodules; and (b) pollen-style interactions are mediated by wall chemistry.
     
11. economic products - cell walls are important for products such as paper, wood, fiber, energy, shelter, and even roughage in our diet.
    

II. Wall Components - Chemistry
    The main ingredient in cell walls are polysaccharides (or complex carbohydrates or complex sugars) which are built from monosaccharides (or simple sugars).  Eleven different monosaccharides are common in these polysaccharides including glucose and galactose. Carbohydrates are good building blocks because they can produce a nearly infinite variety of structures.  There are a variety of other components in the wall including protein, and lignin. Let's look at these wall components in more detail:

A. Cellulose
   β1,4-glucan (structure provided in class). Made of as many as 25,000 individual glucose molecules. Every other molecule (called residues) is "upside down". Cellobiose (glucose-glucose disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with itself (intra-molecular H-bonds) and with other cellulose chains (inter-molecular H-bonds). A cellulose chain will form hydrogen bonds with about 36 other chains to yield a microfibril. This is somewhat analogous to the formation of a thick rope from thin fibers. Microfibrils are 5-12 nm wide and give the wall strength - they have a tensile strength equivalent to steel. Some regions of the microfibrils are highly crystalline while others are more "amorphous". 

B. Cross-linking glycans (=Hemicellulose)
    Diverse group of carbohydrates that used to be called hemicellulose.  Characterized by being soluble in strong alkali. They are linear (straight), flat, with a β-1,4 backbone and relatively short side chains. Two common types include xyloglucans and glucuronarabinoxylans. Other less common ones include glucomannans, galactoglucomannans, and galactomannans.  The main feature of this group is that they don’t aggregate with themselves - in other words, they don’t form microfibrils. However, they form hydrogen bonds with cellulose and hence the reason they are called "cross-linking glycans". There may be a fucose sugar at the end of the side chains which may help keep the molecules planar by interacting with other regions of the chain.

C. Pectic polysaccharides
    These are extracted from the wall with hot water or dilute acid or calcium chelators (like EDTA).  They are the easiest constituents to remove from the wall. They form gels (i.e., used in jelly making). They are also a diverse group of polysaccharides and are particularly rich in galacturonic acid (galacturonans = pectic acids).   They are polymers of primarily β 1,4 galacturonans (=polygalacturonans) are called homogalacturons (HGA) and are particularly common.  These are helical in shape. Divalent cations, like calcium, also form cross-linkages to join adjacent polymers creating a gel.  Pectic polysaccharides can also be cross-linked by dihydrocinnamic or diferulic acids.  The HGA's (galacturonans) are  initially secreted from the golgi as methylated polymers; the methyl groups are removed by pectin methylesterase to initiate calcium binding.

    Other pectic acids include Rhamnogalacturonan II (RGII) which features rhamnose and galacturonic acid in combination with a large diversity of other sugars in varying linkages.  Dimers of RGII can be cross-linked by boron atoms linked to apiose sugars in a side chain.  

    Although most pectic polysaccharides are acidic, others are composed of neutral sugars including arabinans and galactans.  The pectic polysaccharides serve a variety of functions including determining wall porosity, providing a charged wall surface for cell-cell adhesion - or in other words gluing cells together (i.e,. middle lamella), cell-cell recognition, pathogen recognition and others.

D. Protein
    Wall proteins are typically glycoproteins (polypeptide backbone with carbohydrate side chains). The proteins are particularly rich in the amino acids hydroxyproline (hydroxyproline-rich glycoprotein, HPRG), proline (proline-rich protein, PRP), and glycine (glycine-rich protein, GRP).  These proteins form rods (HRGP, PRP) or beta-pleated sheets (GRP).  Extensin is a well-studied HRGP.   HRGP is induced by wounding and pathogen attack. The wall proteins also have a structural role since: (1) the amino acids are characteristic of other structural proteins such as collagen; and (2) to extract the protein from the wall requires destructive conditions. Protein appears to be cross-linked to pectic substances and may have sites for lignification. The proteins may serve as the scaffolding used to construct the other wall components. 

    Another group of wall proteins are heavily glycosylated with arabinose and galactose. These arabinogalactan proteins, or AGP's, seem to be tissue specific and may function in cell signaling. They may be important in embryogenesis and growth and guidance of the pollen tube.

E. Lignin
    Polymer of phenolics, especially phenylpropanoids. Lignin is primarily a strengthening agent in the wall. It also resists fungal/pathogen attack.

F. Suberin, wax, cutin
    A variety of lipids are associated with the wall for strength and waterproofing.

G. Water
    The wall is largely hydrated and comprised of between 75-80% water. This is responsible for some of the wall properties. For example, hydrated walls have greater flexibility and extensibility than non-hydrated walls.

III. Morphology of the Cell Wall - there are three major regions of the wall:

1. Middle lamella - outermost layer, glue that binds adjacent cells, composed primarily of pectic polysaccharides.
   
2. Primary wall - wall deposited by cells before and during active growth. The primary wall of cultured sycamore cells is comprised of pectic polysaccharides (ca. 30%), cross-linking glycans (hemicellulose; ca 25%), cellulose (15-30%) and protein (ca. 20%) (see Darvill et al, 1980). The actual content of the wall components varies with species and age.  All plant cells have a middle lamella and primary wall.
   
3. Secondary Wall - some cells deposit additional layers inside the primary wall. This occurs after growth stops or when the cells begins to differentiate (specialize). The secondary wall is mainly for support and is comprised primarily of cellulose and lignin. Often can distinguish distinct layers, S1, S2 and S3 - which differ in the orientation, or direction, of the cellulose microfibrils.

IV. Tire analogy for the cell wall
    The wall is similar to a tire that has a series of steel belts or cords embedded in an amorphous matrix of rubber. In the plant cell wall, the "cords" are analogous to the cellulose microfibrils and they provide the structural strength of the wall. The matrix of the wall is analogous to the rubber in the tire and is comprised of non-cellulosic wall components. How are the various wall polymers arranged?  It appears that:

1. cross-linking glycans (hemicellulosic polysaccharides) are hydrogen bonded to the cellulose microfibrils
2. cross-linking glycans may also be entrapped inside cellulose microfibrils as they form
3. the different types of pectic polysaccharides are covalently bonded to one another
4. calcium bridges link pectic acids
5. connections between the protein and other wall polymers are still not clear
6. pectic polysaccharides and cross-linking glycans interact 
7. cross-linking glycans are linked by ferulic acid bridges or boron

V. Wall Formation
    The cell wall is made during cell division when the cell plate is formed between daughter cell nuclei. The cell plate forms from a series of vesicles produced by the golgi apparatus. The vesicles migrate along the cytoskeleton and move to the cell equator. The vesicles coalesce and dump their contents. The membranes of the vesicle become the new cell membrane. The golgi synthesizes the non-cellulosic polysaccharides. At first, the golgi vesicles contain mostly pectic polysaccharides that are used to build the middle lamella. As the wall is deposited, other non-cellulosic polysaccharides are made in the golgi and transported to the growing wall.

    Cellulose is made at the cell surface. The process is catalyzed by the enzyme cellulose synthase that occurs in a rosette complex in the membrane. Cellulose synthase, which is initially made in by the ribosomes (rough ER) and move from the ER → vesicles →  golgi →  vesicle →  cell membrane.  The enzyme apparently has two catalytic sites that transfer two glucoses at a time (i.e., cellobiose) from UDP-glucose to the growing cellulose chain.  Sucrose may supply the glucose that binds to the UDP.   Wall protein is presumably incorporated into the wall in a similar fashion.

    Remember that the wall is made from the outside in. Thus, as the wall gets thicker the lumen (space within the wall) gets smaller.

    Exactly how the wall components join together to form the wall once they are in place is not completely understood. Two methods seem likely: 

1. self assembly. This means that the wall components spontaneously aggregate; and
    
2. enzymatic assembly – various enzymatic reactions (XET) are designed for wall assembly. For example, one group of enzymes "stitches" xylans together in the wall to form long chains.  Oxidases may catalyze additional cross-linking between wall components and pectin methyl esterase may play an important role (see below).

VI. Strong Wall/Cell Expansion Paradox (don’t you just love a good paradox?)
    How can the wall be strong (it must withstand pressures of 100 MPa!), yet still allow for expansion? Good question, eh? The answer requires that the wall:

A. Be capable of expansion
    In other words, only cells with primary walls are capable of growth since the formation of the secondary wall precludes further expansion of the cell. The sequence of microfibril orientation changes during development. Initially the microfibrils are laid down somewhat randomly (isotropically). Such a cell can expand in any direction. As the cell matures, most microfibrils are laid down laterally, like the hoops of a barrel, which restricts lateral growth but permits growth in length.  As the cell elongates the microfibrils take on an overlapping cross-hatched pattern, similar to fiberglass. This occurs because the cell expands like a slinky - the width of the cell doesn't change by the microfibrils become aligned in the direction of growth just like the spring. This overlapping of microfibrils, which is strong and lightweight, prohibits further expansion.  

    But, what determines the orientation of the microfibrils? They are correlated with the direction of the microtubules in the cell. Evidence: treating a cell with colchicine or oryzalin (which inhibit microtubule formation) destroys the orientation of the microfibrils. The microtubules apparently direct the cellulose synthesizing enzymes to the plasma membrane.

    In addition to cellulose microfibril orientation, mature walls apparently loose their ability to expand because the wall components become resistant to loosening-activities. This would occur if there were increased cross-linking between wall components during maturation. This would result from:

1. producing wall polysaccharides in a form that makes tighter complexes with cellulose or other materials
2. increasing the lignin in the wall would increase cross-links between polymers
3. de-esterifying the pectic acids would increase calcium bridges;

B. Loosening (or Stress relaxation) the wall at the appropriate time
    Even though the microfibrils may be in the proper position to permit loosening, the wall is still rather strong. Recall that our wall model proposed strong (covalent) and weak (hydrogen bonds) links between the wall components. When the wall is loosened, weak bonds are temporarily broken to allow the wall components to slide or creep past one another. So, how is the wall temporarily loosened?

1. Protons are the primary wall loosening factor (Acid Growth Hypothesis). This idea was first proposed by David Rayle and R. Cleland in 1970. Some evidence:

• acid buffers stimulate elongation and rapid responses 5-15 min even in non-living tissues (Evans,1974);
• acid secretion is associated with sites of cell elongation (see Evans & Mulkey, 1981)
• Fusicoccin, a diterpene glycoside extracted from a fungus, stimulates proton secretion (activates a H⁺/K⁺ pump) and stimulates elongation.

2. Mechanism of proton action: Protons stimulate wall loosening by:

• disrupting acid-labile bonds such as H-bonds and calcium bridges; and
  
• enhancing the activity of enzymes that break wall cross-links including H-bonds and calcium bridges. Evidence for the enzyme involvement includes: (1) when primary walls are heated or treated with protein denaturing agents they can't be "loosened" by acid; and (2) adding proteins extracted from growing walls to heat-treated walls restores the acid response.
  
      Expansins – appear to be the primary wall-loosening enzymes. This class of proteins are activated by low pH and break the hydrogen bonds between cellulose and the cross-linking glycans. Other candidates for enzymes involved include: (1) pectin methyl esterase which would break the calcium bridges between pectins by esterifying the carboxyl groups; and (2) hydrolases – which would hydrolyze the cross-linking glycans (hemicelluloses).  For example, xyloglucan endotransglycosylase (XET) has been shown to cleave cross-linking glycans that could allow slippage of the wall components

 3. The acid effect is induced by indole-3-acetic acid (IAA, auxin), one of the major plant hormones. IAA stimulates proton excretion and cell growth/elongation.  Evidence:

• peeled coleoptiles + IAA → medium acidic; peeled coleoptiles + water → not acidic; and
• flooding auxin-treated tissue with neutral buffers prevents the growth response.

4. Mechanism of Auxin Action – How does auxin stimulate proton excretion and wall elongation?  There are two ideas:

Hypothesis 1:  Auxin activates pre-existing H⁺-ATPase pump proteins in the cell membrane. These proteins transport protons from the protoplast into the wall. Auxin probably first binds to a receptor molecule and this complex then actives the pump. This process is active - thus the pump requires ATP. Evidence: ATP stimulated acidification is observed soon after auxin treatment.

Hypothesis 2: Auxin stimulates transcription and translation. Transcription/translation (protein synthesis) would be required to produce proton pump proteins (a wonderful alliteration), respiratory enzymes to provide ATP to power the process; and even enzymes for the synthesis of wall components and cell solutes (see C. & D. below). Evidence for the involvement of transcription/translation:

• Nooden (1968) found that artichoke disks increased in size when incubated with IAA but that the addition of antimycin (a protein synthesis inhibitor) prevented this response;
• soybean hypocotyls incubated with 2,4-D (an analog of IAA) produce at least 3 new polypeptides within three hours (Zurfluh & Guilfoyle, 1980);
• in vitro translation of mRNA occurs within 15 minutes of IAA treatment
• The proton effect is short-lived. Cell elongation stops 30-60 minutes after acidification. Continuous elongation requires longer term metabolic changes such as protein synthesis.

C. Wall synthesis occurs
    As the cell grows, wall synthesis needs to occur. Think about the color of a balloon as it is blown up - it gets lighter in color as the balloon gets larger because the thickness of the balloon decreases as it expands and stretches. Using this logic, we expect that plant cells should become thinner as they expand. Right? Wrong - cell walls remain a relatively uniform thickness throughout cell growth. Thus, we can conclude that new wall material must be made during cell elongation.

D. Enhanced solute synthesis
    The solute concentration of the cell remains constant during cell enlargement. This suggests that solutes are being synthesized since the volume of the cell is increasing. Maintaining a high solute concentration is necessary to allow for water uptake.

E.  Lock wall in place after expansion is complete 
    Once wall elongation is completed, the cell needs to "lock it" in place.  This likely happens as the temporary bonds that were broken reform, and due to increased interactions (including enzymatic) between wall molecules.

F.  Water Uptake/Pressure

Web Sites:

• Carpita, N, McCann (2000) Cell Walls. Chapter 2. In Biochemistry & Molecular Biology of Plants.  Buchanan, BB, Gruissem W, Jones, RL. eds. American Society of Plant Biology, Beltsville, MD.  This is a great article!

References

• Albersheim, P. 1975. The Walls of Growing Plant Cells. Sci. Amer. 232: 81-95.
• Albersheim, P. 1985. Oligosaccharins. Sci. Amer. 253: 58.
• Brett, C. T. and J.R.Hillman. 1985. Biochemistry of Plant Cell Walls. Cambridge University Press, NY.
• Brett, C. & K. Waldron. 1996. Physiology and Biochemistry of Plant Cell Walls. 2nd edn. Chapman & Hall, NY.
• Cosgrove, D. 1986. Cell growth. ARPP 37: 377
• Delmer, D. 1987. Cellulose biosynthesis. ARPP 38: 259
• Fry, S.C. 1989. Dissecting the complexity of the plant cell wall. Plants Today 2: 126-132.
• Mulkey, T.J., K.M. Kuzmanoff and M.L. Evans. 1981. The agar-dye method for visualizing acid efflux paterns during tropistic curvatures. What's New in Plant Physiol. 12:9-12.
• Preston, R.D. 1979. Polysaccharide conformation and cell wall formation. ARPP 30:50.
• Taiz, L. 1984. Plant cell expansion. ARPP 35: 585-647
• Ruszkowski, Martha - check this link for the Ukrainian translation by M Ruszkowski.

Last updated:  09/30/2011     � Copyright  by SG Saupe