Lecture 8 - Plant cell walls

Question 1

What are the two general types of cell walls?

Answer 1

• Primary cell walls
  
  • thin
  • strong
  • flexible to allow growth
  • all plant cells
• Secondary cell walls
  
  • thick
  • stronger
  • durable to support stems and resist digestion
  • only some plant cells
  • aquire after stop growing to give strength

Question 2

What is the importance of plant cell walls in plants?

Answer 2

• Mechanical support to cells and to the organism
• Cell to cell adhesion
• Cell to cell communication
• Transport
• Interactions with pathogens
• Cell expansion - growth and morphogenesis

Question 3

How do plants grow quickly?

Answer 3

• By inflating their cells
• Large vacuoles in the cell can increase by 100-1000 fold in size

Question 4

What occurs when cells are under pressure?

Answer 4

• Cells can expand at a rate of 10-20%/hour (vacuole)
• Typical plant cell has turgor pressure of around 5atm - 75 psi (twice the pressure of a car tyre)
• Therefore cell wall must be strong
• get extension under stress
• dynamic control

Question 5

Give an example of cell expansion under stress

Answer 5

In dark conditions get etiolated seedlings

Under dark conditions cells are plastic

When light is shone upon they become rigid

Question 6

What are the features of primary cell walls?

Answer 6

• Strong enough to withstand stress induced by turgor
• Flexible enough to allow growth
• Have inherent elasticity - if remove turgor pressure cell will contract by around 5%
• Have dynamic plasticity - able to expand in a controlled manner driven by turgor to affect cell expansion

Question 7

What is the composition of the primary cell wall?

Answer 7

• flexible but strong fibre composite material
• 1/3 dry weight is cellulose microfibrils (rigid with a higher tensile strength than steel)
• 1/3 hemicellulose
• 1/3 pectins
• high water content (hemicelluse is very soluble-makes structure flexible)
• Little or no covelent bonds between polymer groups

Question 8

What is hemicellulose?

Answer 8

Neutral polysaccharides that hydrogen bond to cellulose microfibrils and bind them to one another to form a cohesive network

Question 9

What are pectins?

Answer 9

Acidic polysaccharides that interpenetrate the hemicellulose network and provide a strong hydration potential to the wall

Question 10

What is cellulose?

Answer 10

• Most abundant biological polymer on earth
• 30% of wall dry weight
• Stable microfibrils - hard to digest
• composed of linear (1,4)-beta-D-glucans
• typically insoluble

Question 11

What are cellulose microfibrils?

Answer 11

• composed of many glucan chains associated in crystalline arrays to produce long microfibrils
• held together by hydrogen bonds
• 20-30 glucan chains in a microfibril
• very strong and rigid
• can measure many micrometers
• greater tensile strength than steel

Question 12

What is the process of formation of a cellulose microfibril?

Answer 12

• Made by protein somplexes in plasma membrane which have a hexameric conformation (rosette)
• Rosette: 6 large protein complex bound into macro protein complex in the membrane each with an active site. Cellulose synthase complex - they produce a glucan chain in syncrony
• glucan chain made of linear (1,4)-beta-D-glucans
• comes together to form sub fibrils -> microfibril -> travel through the membrane and are displaced in the membrane

Question 13

What are hemicelluloses?

Answer 13

• associated with cellulose microfibrils by extensive hydrogen bonding to form main structural network of the wall
• long polysaccharides based on (1, 4)-beta-D-glucans or xylans (chemically identical)
• Have substantial side chains (made in the golgi, more soluble) that prevent the formation of crystalline structures

Question 14

What is the structure of xyloglucans?

Answer 14

Glc backbone

Side chains of Xyl, or Xly and Gal, or Xly and Gal and Fuc

Question 15

What are xyloglucans?

Answer 15

One of the most common hemicelluloses in the cell wall

Question 16

How do xyloglucans bind microfibrils?

Answer 16

• Some regions of hydrogen chain can H bond to microfibrils
• Get regions where binds to two microfibrils binding them together
• Results in a complex coated in hemicellulose
• Prevents structure from sticking to other microfibrils
• Holds composite together and holds individual fibrils apart

Question 17

What is the paradoxical role of xyloglucans?

Answer 17

Act as both plasticisers and tethers for cellulose microfibrils

Crystalline cellulose coated with paracrystaline cellulose and a hemicellulose mesh

Question 18

How is cell wall expansion under dynamic control?

Answer 18

Enzymes/proteins in the cell wall which have catalytic function to change rheology of cell walls leading to expansion

e.g. expansins induce expansion of cell walls

Question 19

How was the role of expansins discovered?

Answer 19

1. Seedlings growing in dark grow upwards rapidly
2. Only top part growing
3. Take top cells and attach to a device which exerts a weight on the cells
4. shows that extension of the cell walls has a distinct pH dependence
5. at neutral pH they are functioning but don’t extend
6. at acididc pH they rapidly extend
7. acid extension can be lost if cell walls are proteolysed
8. Isolated proteins from cell walls and recomplemented to inactivated cell walls and got reactivation of expansion

expansins

Question 20

What is the action of expansin?

Answer 20

• induce extension without hydrolysis of substrates
• bind at an interface between cellulose microfibrils and crosslinking xyloglucans
• disrupt hydrogen bonds that hold polymers together

Question 21

How can the plant regulate the activity of expansins?

Answer 21

• Expansins are only active at low pH
• plants can regulate expansin by changing pH in cell wall
• proton pumps - acidify by pumping out protons, in light pH is less acidic and wall becomes rigid and unexpandable

Question 22

What is pectin?

Answer 22

• comprises roughly 30% of primary cell walls
• makes independent coextensive network with cellulose/hemicellulose
• wall adhesion important for wall elasticity
• not found in secondary cell walls
• less abundant in the walls of grasses
• acidic polysaccharides (have acidic nature)

Question 23

What is homogalacturonan?

Answer 23

• A major pectin component
• doesn’t exist in isolation - secreted with 60% carboxyl hidden by methyl esters that are associated
• secreted in methylated state into cell wall
• Enzymes in the cell wall remove methyl groups
• degree of HGA methylation determines acidity, ability to cross link and susceptibility to hydrolases
• alpha 1,4 linked polymer of galacturonic acid HGA
• sugar with carboxyl groups which combine to divelent cations forming crosslinks between two ions (typically calcium ions)
• found at junctions between cells
• key to adhesion

Question 24

What is rhammogalacturonan1?

Answer 24

A major component of pectin important in wall eleasticity

Has a galacturonic acid backbone interspersed with arabinan residues

Can carry side chains (5 arabinan, 4-galactan, type I arabinogalactan)

Question 25

How are HGA and RGA1 linked?

Answer 25

covelelently

Question 26

What determines the stiffness/flexibility of the cell wall?

Answer 26

The ratio of RG1 and HGA

Question 27

What is the role of rhamnogalacturonan1 in stomatal guard cell walls?

Answer 27

* Play a role in the elasticity of stomatal guard cell walls * Modulates the appeture of stomatal guard cell walls, which is controlled by altering the turgor pressure of cells * Two guard cells are anchored strongly to each other with the inner edge having a stronger cell wall than the outer edge * As cells increase in pressure (up to 750psi during opening) pore opens - important for regulating water loss during photosynthesis * Inner cell walls are 20 times thicker than normal cell wall * High (70%) elasticity compared to the 5% in most cell walls

Question 28

What is the function of arabinose side chains in rhamnogalacturonan1?

Answer 28

Keep homogalacturonan chains apart preventing crosslinking Maintains pectin fluidity and wall flexibility

Question 29

How was the role of rhamnogalacturonan1 in guard cells discovered?

Answer 29

Removed different components of the cell wall and guard cells and looked at the ability of stomata to open and close If removed the hairy regions of rhamnogalacturonan1 the stomatal regions can no longer open If remove whilst they are open, can no longer close Completely lose elasticity Hairy regions: Rhamnogalacturonan1

Question 30

What is RGA-II?

Answer 30

* Highly conserved very complex structure * requires more than 20 enzymes to make it * dimerises in the presence of boron - critical to plant growth * Backbone of homogalacturonan with very complex side chains * Structure completely conserved within all land plants * If structure is distructed by knocking out linkages, no longer forms dimer with boron which is lethal * If only weakly bind boron = dwarf plant

Question 31

What are the features of secondary cell walls?

Answer 31

* depositied to reinforce certain cells after cell has finished growing * depositied inside primary cell walls * thick, strong and rigid * have little elasticity and extensibility * provide permenent structures for the plant

Question 32

What is the composition of the secondary cell wall?

Answer 32

* 1/3 dry weight cellulose microfibrils - rigid with a higher tensile strength than steel * 1/3 hemicellulose - different to primary as typically xylose * 1/3 lignin * very low water content * many covelent links between polymer networks * no pectin

Question 33

What is lignin?

Answer 33

* A polyphenol that impregnantes and seals the cellulose-hemicellulose network making it rigid and hydrophobic * second most abundant biological polymer * major component of secondary walls * No repeat structureso hard to break down by enzymes

Question 34

How does lignin get incorperated into the secondary cell wall cellulose/hemicellulose network?

Answer 34

* Secreted as monomers into the wall and polymerised to form strong/resistance encapsidation 1. monolignols polymerised in wall to form complex structure 2. oxidatively cross linked using free radicals generated from peroxidases and lacases 3. radialise monomers 4. then when they collide with another they cross link

Question 35

What are woody plant cell walls made of?

Answer 35

* Mostly polysaccharides and lignin * Cellulose hydrogen bound to hemicellulose and lignin associates by being covelently corsslinked to the hemicelluloses

Question 36

What is the importance of primary cell walls in agriculture?

Answer 36

* growth and development - yield, stature etc. * crop protection - first line of perception and defence against pathogens * fruit ripening - softening of fruit involves the breakdown of cell-cell adhesion and wall softening * Abscission and dehiscence - controlled shedding of plant organs by controlled cell wall autolysis * Source of polysaccharides for food and personal care industries e.g. pectins

Question 37

What is the importance of secondary cell walls in industry?

Answer 37

* paper * builiding materials (wood - v. light, good weight to strength ratio) * textiles and composites * Fuel (liquid transportation fuels)

Question 38

Why can we no longer rely on petroleum?

Answer 38

* finite resource * passed peak production * increased global demand driving production in to more extreme sources * cobustion of fossil carbon has doubled CO2 levels since the start of the industrial revolution * which is impacting the environment in unpredictable ways

Question 39

What are the pros and cons of petroleum use?

Answer 39

**Pros** * provides liquid transportation fuels as well as industrial chemicals and materials * available in bulk * cheap * mature industrial conversion **Cons** * Non renewable * finite and diminishing resource * CO2 emitting * hazardous and polluting * unsustainable

Question 40

What are the pros and cons of the use of plant biomass?

Answer 40

**Pros** * potential to provide liquid fuels, chemicals and materials * abundant * renewable * cheap * potentially CO2 neutral **Cons** * ​Nascent conversion technologies * underdeveloped supply chain * sustainability as basis of industrial economy unclear

Question 41

What are the two types of plant biomass?

Answer 41

Lignocellulosic biomass Food - Starch, sucrose, oils (1st generation biofuels)

Question 42

Why is the use of first generation biofuels unsustainable?

Answer 42

Added stress on food security

Question 43

What can be used instead of first generation biofuels?

Answer 43

Instead look at the non food parts of plants, lignoellulistic biomass, wood residues 1. Make biofuels by chemical transformation/non chemical 2. have to break polysaccharides in cell walls 3. into simple sugars that can be fermented 4. secondary cell walls hard to hydrolyse (expensive)

Question 44

Why is lignocellulosic pant biomass a potential replacement for petroleum?

Answer 44

Source of energy rich carbon compounds * crop residues (straw, husks - more polysaccharides in the straw than the starchy grains) * timber residues * waste * dedicated biomass crops (willow, miscanthus)

Question 45

Why is the use of dedicated biomass crops not good?

Answer 45

Land use change Food crop may be displaced

Question 46

What are the features of lignocellulosic plant biomass?

Answer 46

* strong fibre composite material * durable * rich in polysaccharides * lignin is problematic

Question 47

What approaches are there to improve digestibility?

Answer 47

* Make the cell walls more digestible without compromising field performance * produce more effective enzymes and pretreatments for conversion

Question 48

What was generated to meaure the digestibility of crops?

Answer 48

* Developed a system to measure the cell wall digestibility of large scale populations of mutants/crops * The main cost in biofuels is the coversion to sugars, need a lot of heat/chemicals to pretreat ligniocellulose to open up the structure for enzymes 1. Automated sample grinding and weighing on one side and an automated digestion assay on the other 2. 96 well plates into the loading platform 3. Take the biomass and give a heat pretreatment by acid/alkaline additions 4. Rinse and then add commercial cellulose cpcktail to digest material 5. Incubate in ovens 6. pipette out aliquots from each well 7. colometric assays to tell how much sugar is released

Question 49

What was found using the cell wall disgestibility in Brachypodium digestibility mutants and how?

Answer 49

1. Mutated brachypodium population and screen for plants showing increased disgestibility, ignored plants that were dwarfed 2. Isolated 12 inheritable mutations showing mendelian segregation for increased digestibility (sac1-12) 3. Analysed the composition of the muatants, 1/2 were lignin mutants, others not obvious 4. Characterised these mutants * One was a mutant in a transferase gene that is required in the synthesis of menolignols. A pathway that produces 3 different menolignols to make up the lignin polymer * When get HCT gene knocked out get an increase in digestibility 5. Other groups had shown that when knock out the HCT gene in dicot angiosperms (tobacco, alfalfa, arabidopsis) get dwarfed plants. arabidopsis = lethal * grasses have multiple copies of HCT, dicots only have one or two and arabidopsis has 1 * brachypodium has 3 - when 1 is knocked out get increased digestibility but no effect on crop 6. Second mapping also identified that the sac1 mutant has alesion in the gene encoding glycosyltransferase GT61 protein that is involved in making hemicelluloses in grasses * think that sac1 gene is an ortholog of a gene XAX1 in rice - both have less xylose in hemicellulose and less ferulic acid

Question 50

What are the features of ferulic acid that make cells walls less digestible?

Answer 50

* cross links polymers in cell wals to each other * Hemicellulose to hemicellulose linkage * hemicellulose to lignin linkage * Some arabinyl side chains have ferulic acid on -\> oxidising cross link between separate xylan polymers * only grass xylans have feruloyl esters that cross link them to lignin * Also form cross links to lignin polymer * act as an interface between one polymer system and another to form covelent linkages where before there would be held together by hydrogen bonds

Question 51

How do dicot xylans crosslink to lignin?

Answer 51

Potentially through metylglucuronic acid residues

Question 52

What was shown through association genetics in Barley? (Waugh and Halpin, Dundee)

Answer 52

* high density single nucleotide polymorphism markers defined in 850 elite barley cultivars from around the world * phenotypic screening of these cultivars can then be used with SNP association mapping to define the quantitative trait loci at high resolution * quantitative traits (such as digestibility) are defined by many genes, QTL analysis allows the identification of major genetic contributors to a trait * 640 genotypes of elite spring barley were ranked by the yield of sugar released in a simple saccharification assay, had 5 replicate plants per genotype (with 3 technical assay replicates per plant) * QWAS identified several QTL for digestibility in barley straw

Question 53

Why are GWAS studies often done in rice?

Answer 53

* the wheat genome is massive and hexaploid and has not been fully sequenced * barley has a very large genome that has not been fully assembled but it is diploid and can serve as a model for wheat * rice has the smallest genome of any cereal (only two times arabidopsis) and is the second largest produced crop in the world

Question 54

Why might rice straw be good to use for biofuel?

Answer 54

* Vietnam produces more then 60million tons of rice straw and most is burned but could be used for energy for biofuels * unusually high levels of silica (up to 10%) make RS unpalatable to aninmals and impractical for combustion to produce bioelectricity * every year hundreds of millions of tonnes are burned in the field globally * burning causes: * air pollution responsible for large numbers of premature deaths * reduced crop yeild from shading and generation of a tropospheric ozone * global warming from atmospheric black carbon * could improve straw quality for biofuel and animal feed by increasing the digestibility and decreasing Si content

Question 55

How was a rice diversity panel produced for GWAS studies?

Answer 55

1. Assembled diversity of 180 rice accessions with the vietnam academy of agricultural sciences 2. mapped SNPs using genotype by sequencing methods 3. measured digestibility on robotic platform 4. silica content using x-ray fluoresence 5. identified 20 regions on the manhattan plot for QTL regions of interest 6. found a correlation between digestibility and silicon content (high silicon low digestibility) 7. Manhattan plot of the GWAS on silicon content - maps onto digestibility QTL gene that makes covelent linkages between silicon

Question 56

What future work is likely to be done for the rice digestibility work?

Answer 56

* identify QTL for straw digestibility, silica content and drought resistance to generate markers for breeding * make bi-parental crosses to establish recombinant inbred lines in order to further resolve QTL for straw digestibility and silica content * identify candidate genes responsible for the observed variation in digestibility and silica content at selected QTL * use straw from lines identified to have high digestibility in demonstration activites for animal feed and biofuel applications * carry out dissemination activities with local stakeholders in order to demonstrate the benefits of making use of rice straw and avoiding biomass burning