Plant Science:
Revision Notes

Compiled as a third-year student at the University of Edinburgh, based on the information given in lectures.

Photosynthesis

Radioactive tracing of biochemical pathways

• Melvin Calvin fed carbon-14 to photosynthesising algae (Chlorella)
• ¹⁴C --> ¹⁴N + e^- (beta particle), half-life 5700 years
• Radioactivity detected by autoradiography, Geiger counter, or scintillation counting
• Metabolites can be separated and identified by co-chromatography and enzymic dissection

Carbon fixation by RuBisCO

• Ribulose 1,5-bisphosphate + CO₂ --> two molecules of 3-phosphoglyceric acid (PGA)
• Catalysed by a lot of RuBisCO (ribulose bisphosphate carboxylase oxygenase) enzyme, with 8 large subunits (encoded in chloroplast genome) and 8 small subunits (encoded in nucleus)
• Carboxylase enzymes (reacting CO₂ with organic compounds) are common - what is special about RuBisCO is that it produces a sugar-acid that can easily be converted to sugar
• Reaction is exergonic ('downhill')
• Reaction is very slow (V_max = 2 µmol/min/mg, compared with >100 for most enzymes), perhaps because substrate concentration is low: RuBisCO acts on a rare intermediate that is in equilibrium with RuBP, and CO₂ is in equilibrium with HCO₃^-
• Carbonic anhydrase speeds up HCO₃^- --> CO₂ reaction, but mutants manage fine without it

Fate of PGA

• Phosphoglycerate kinase adds phosphate:
  3-phosphoglyceric acid + ATP --> 1,3-bisphosphoglyceric acid
• Phosphoglyceraldehyde dehydrogenase (in reverse) removes phosphate and adds hydrogen:
  1,3-bisphosphoglyceric acid + NADPH+H⁺ --> 3-phosphoglyceraldehyde + P_i
• ATP and NADPH+H⁺ come from light reactions of photosynthesis
• Out of every six phosphoglyceraldehyde molecules produced, five are recycled to regenerate ribulose bisphosphate; the remaining molecule is 'profit'

Import and export of substances from chloroplast

• Chloroplast contents can be analysed by centrifuging chloroplast suspension in a layer above a layer of acid, separated by silicone oil - chloroplasts are forced through oil and burst in acid
• Chloroplast membranes are freely permeable to water and CO₂
• In experiments, many small compounds (e.g. fructose, RuBP) are taken up by chloroplasts only in small amounts - they pass through outer membrane but not inner membrane
• 3PGA, G3P and DHAP are moved by phosphate translocator, in exchange for P_i
• Chloroplast inner membrane contains translocators for some other small organic compounds

Starch synthesis

• By starch phosphorylase (working in reverse):
  glucose-1-phosphate + (glucose)_n --> (glucose)_n+1 + P_i
• By starch synthase - this turns out to be the more important mechanism:
  ADP-glucose + (glucose)_n --> (glucose)_n+1 + ADP
• Production of ADP-glucose from glucose-1-phosphate, by ADP-glucose pyrophosphorylase (which is activated by PGA and inhibited by P_i), is the rate-limiting step in starch synthesis

Sucrose synthesis

• Sucrose is unusual because it has no reducing group - reducing groups of glucose and fructose components are both tied up in glycosidic bond, which has an unusually high energy
• 3-phosphoglyceraldehyde <--> dihydroxyacetone-phosphate (DHAP)   (catalysed by isomerase)
• 3-phosphoglyceraldehyde + DHAP --> fructose-1,6-bisphosphate    (catalysed by aldolase)
• fructose-1,6-bisphosphate --> fructose-6-phosphate   (catalysed by fructose 1,6-bisphosphatase - FBPase); this reaction can be reversed by PFP (pyrophosphate : fructose 6-phosphate phosphotransferase) - a potential futile cycle
• fructose-6-P --> glucose-6-P --> glucose-1-P --> UDP-glucose
• Sucrose phosphate synthase catalyses a transglycosylation reaction:
  UDP-glucose + fructose-6-phosphate --> UDP + sucrose-6'-phosphate
• The phosphate is then removed by sucrose phosphatase in an irreversible reaction:
  sucrose-6'-phosphate + H₂O --> sucrose + P_i    (delta-G⁰' very negative)
• Sucrose could also potentially be produced by sucrose synthase, but this enzyme does not occur in adequate quantities and the reaction is worryingly reversible:
  UDP-glucose + fructose <--> UDP + sucrose
• Sucrose can be broken down by sucrose synthase (working in reverse) or by invertase

Control of sucrose synthesis

• P_i (which accumulates in cytosol at times of low G3P production) inhibits fructose 1,6-bisphosphatase and sucrose phosphate synthase, reducing sucrose production
• AMP (which is produced from ADP under starvation conditions) strongly inhibits fructose 1,6-bisphosphatase, reducing sucrose production
• Signalling molecule fructose-2,6-bisphosphate inhibits conversion of fructose-1,6-bisphosphate to fructose-6-phosphate (inhibiting FBPase and activating PFP), reducing sucrose production
• Fructose-2,6-bisphosphate is produced from fructose-6-phosphate; P_i and fructose-6-phosphate promote its formation and inhibit its breakdown
• Photosynthetic products (C₃P₁ compounds) inhibit fructose-2,6-bisphosphate production
• Excess glucose-6-phosphate (a sign of plenty) activates sucrose phosphate synthase

Photorespiration

• Plants respire faster in light than in the dark, due to photorespiration
• Fuel for photorespiration is glycollate (adding glycollate to leaves increases photorespiration; glycollate oxidase inhibitors prevent it; giving ¹⁴CO₂ to leaves produces ¹⁴C-glycollate)
• Production of glycollate is light dependent, promoted by high O₂, and decreased by high CO₂ - O₂ and CO₂ compete as substrates for RuBisCO
• ribulose bisphosphate + O₂ --> PGA + phosphoglycollate   (catalysed by RuBisCO)
• In chloroplast:
     phosphoglycollate + H₂O --> glycollate + P_i
• Glycollate is exported from chloroplast by glycollate translocator and enters peroxisome
• In peroxisome:
  glycollate + O₂ --> glyoxylate + H₂O₂   (catalysed by glycollate oxidase)
  2H₂O₂ --> 2H₂O + O₂   (catalysed by catalase)
  glyoxylate + an amino acid --> glycine + other compound   (catalysed by transaminase)
• In mitochondrion:
  2 × glycine + H₂O + NAD⁺ --> serine + CO₂ + NH₃ + NADH+H⁺  (catalysed by glycine synthase and serine hydroxymethyltransferase)
• Back in peroxisome:
  serine + other compound --> hydroxypyruvate + an amino acid   (catalysed by transaminase)
  hydroxypyruvate + NADH+H⁺ --> glycerate + NAD⁺   (catalysed by glycerate dehydrogenase)
• Back in chloroplast:
  glycerate + ATP --> 3-phosphoglycerate (PGA) + ADP   (catalysed by glycerate kinase)
• Photorespiration may be a 'metabolic mistake' (photosynthesis evolved at a time of low atmospheric [O₂]), it may have a protective role (dissipating excess energy), or it may be a pathway of glycine and serine synthesis (but these are produced in excessive quantities)
• In light, in the absence of O₂ or CO₂, photosynthetic pigments are damaged
• Plants missing any of the enzymes involved in photorespiration get ill
• Recent increases in atmospheric [CO₂] should reduce photorespiration and improve yields

Plant water relations and nutrient uptake

Water movement

• Movement of water can be apoplastic (around cells), symplastic (through plasmodesmata linking cells) or transcellular (through cells, in and out of membranes)
• Rate of diffusion is proportional to concentration difference / distance (Fick's law)
• Small molecules diffuse faster than large ones
• Bulk flow is the mechanism of transport in xylem and phloem - less resistance than diffusion
• Rate of flow is strongly dependent on tube radius:   F is proportional to r⁴
• Hydrogen-bonding in water results in high surface tension, adhesion, cohesion and capillarity
• Water flows from areas of high water potential to areas of low water potential
• water potential (y_w) = solute potential (y_s) + hydrostatic potential (y_p) + gravity potential (y_g)
• Water has a high tensile strength - it resists breakage of a water column in a capillary
• Gas dissolved in a water column under tension can come out of solution and cause cavitation

Water uptake by plants

• Soil water potential decreases as soil dries
• Plants cannot absorb water if soil potential < -1MPa, resulting in permanent wilting
• Adhesion and cohesion of water to soil particles create forces that resist water removal
• High solute potential in saline soils also makes it harder for roots to take up water
• High solute concentrations in roots, and "pull" from xylem, permit water uptake by plants
• Xylem comprises tracheids (narrow and very long) and vessels (shorter and wider)
• Tracheids help prevent cavitation - perforation plates in them isolate bubbles of gas
• Evaporation creates large water potential differences at leaf surface that drive water movement
• Cellulose microfibrils give leaf cells a large surface area, promoting evaporation

Root structure and function

• Factors affecting crop yield: irrigation, mineral nutrition, disease and pest resistance, stature and life cycle
• Nutrient availability, not photosynthetic ability, generally limits plant productivity
• Functions of roots: anchorage, absorption, conduction, storage, maintaining rhizosphere
• 'Tap root system' consists of long, deep roots (primary root is gravitropic, secondary roots are partially gravitropic, tertiary roots grow in any direction)
• 'Fibrous root system' consists of a shallow mass of roots (either a primary root with a very high branching rate, or a large number of adventitious roots that are partially gravitropic)
• Stele (core of root) is surrounded by an impermeable endodermis (sealed by Casparian strips around cells), preventing water leakage and allowing plants to take up substances selectively
• Root hairs are long (500-1000µm) and ephemeral, existing only close to root apex
• Root hairs are very important in nutrient uptake (>80% of phosphate enters via root hairs)
• Some plants have modified roots that can store nutrition (tubers) or water (large tap roots)
• Roots alter their soil environment (the rhizosphere), secreting a mucigel sheath, to provide lubrication for roots and promote growth of beneficial microorganisms
• Secretion of organic nutrients (highest near root tip due to phloem unloading) and sloughed-off root cells also provide food for soil microorganisms
• 30-60% of photosynthetic carbon goes to roots; 70% of this is lost through rhizo-deposition
• Root exudates are concentrated just behind growing root tip; microorganisms trail behind this
• Roots secrete molecules such as phenolics that attract microorganisms
• Experiments show that wheat plants are more susceptible to disease if soil is sterilised

Enzyme kinetics

• V_max is the reaction velocity of an enzyme at infinitely high substrate concentration
• K_m (Michaelis-Menten) constant is the substrate concentration at which velocity is half V_max
• Michaelis-Menten equation for an idealised enzyme:  V = (V_max [s]) / (K_m + [s]) where V is reaction velocity and [s] is substrate concentration

Electrochemical potential

• Concentration difference of charged ions can establish an electrical potential across membrane
• Conversely, an electrical potential can establish a concentration difference at equilibrium
• Nernst equation:   DE = [RT/ZF][log(C_in/C_out)] where DE is electrical potential, R is universal gas constant, T is temperature (in °K), Z is electrostatic charge of ion, F is Faraday's constant, C_in and C_out are concentrations of ion on either side of membrane
• For a univalent cation at 25°C, this simplifies to:   DE = 59 log(C_out/C_in)

Soil

• Soil contains negatively-charged particles of silicate minerals (derived from underlying rocks)
• Soils often bind cations strongly
• Soil pH determines the solubility of minerals (often most soluble in slightly acidic conditions)
• Most soils are neutral or slightly acidic, due to organic decay and extrusion of H⁺ by roots
• Differential solubility causes nutrients to be concentrated in patches ("jackpots") within soil
• Nitrate permeates easily through soil, and accumulates over water-impermeable layers
• Phosphate moves slowly, due to interactions with soil minerals, and accumulates in topsoil
• Nitrate starvation causes deep root growth; phosphate starvation causes shallow growth

Uptake of phosphate and other mineral nutrients

• Higher plants have many phosphate transporter proteins - symporters that allow plant to take up phosphate by active transport, requiring a potential difference across the membrane
• Roots secrete H⁺, which releases phosphate by cation exchange with soil particles
• Plants secrete phosphatases and nucleases into soil to release organically-bound phosphate
• Mutations in phosphate transport genes:
  Pho1 - defective transport protein, can't transport P from root to shoot --> phosphate starvation
  Pho2 - defective ubiquitin ligase, can't transport P from shoot to root --> P accumulates in shoot
• Lack of phosphate activates phosphate starvation induction (PSI) genes
• Phosphate starvation is detected in shoot, which signals to root (mechanism unknown)
• Responses to lack of phosphate: highly-branched roots with many root hairs, decreased shoot/root ratio, acidification of rhizosphere, metabolic changes to reduce phosphate demand (e.g. substitution of ATP with pyrophosphate), mobilisation of phosphate stored in vacuoles, substitution of phospho-lipids with sulpho-lipids and galacto-lipids.
• Symbiotic associations with mycorrhizae increase soil volume with which plant can interact
• Vascular arbuscular mycorrhizae (VAM) penetrate plant cells, forming arbuscules
• Ectomycorrhizae simply encase roots (making them appear thick)
• Roots secrete organic acids that chelate mineral ions (Fe, Mn), promoting their uptake

Minerals required by plants

• Nitrogen - found in amino acids, nucleic acids, chlorophyll, poly-amines, alkaloids, etc
• Phosphorous - forms high-energy bonds in ATP, sugar phosphates, nucleotides, etc
• Sulphur - found in amino acids methionine and cysteine, vitamins and co-factors; maintains cellular redox poise by glutathione (stabilising sulphide bridges in protein molecules)
• Potassium - maintains cell osmolarity and electro-neutrality, co-factor for enzymes
• Calcium - stabilises cell walls and membranes, co-factor for enzymes, second messenger
• Magnesium - part of chlorophyll, co-factor for enzymes, stabilises structure of ATP
• Silicon - deposited in cell walls to resist pathogens and reduce 'lodging' (breakage by wind)

Plant organelles

Plastids

• Proplastids develop into chloroplasts, etioplasts (if deprived of light), chromoplasts (pigmented, in flowers and fruits) or amyloplasts (starchy, in roots and tubers)
• Chloroplasts are enclosed by a double membrane
• Chloroplasts contain stroma, and granal stacks enclosed by thylakoid membranes
• Light reactions of photosynthesis take place at photosystem 1 and photosystem 2 complexes in thylakoid membranes; H⁺ gradient across thylakoid membrane drives ATP synthesis
• Chloroplasts synthesise amino acids, fatty acids, chlorophyll, haem, isoprenoids (oils, growth regulators, etc), carotenoids, nucleotides, alkaloids and co-enzymes
• Substances can pass between plastids through 'stromules' (stroma-filled tubules)

The plastome

• Plastids contain multiple copies of the 'plastome' - circular DNA, ~150,000 b.p.
• Plastids contains ~100 genes - not all the proteins found in the plastid are encoded here
• Large single copy and small single copy regions separated by two inverted repeat regions
• Plastids genes are responsible for some maternally-inherited plant traits (e.g. variegation)
• Plastid genetics are prokaryote-like: circular and naked genome, 70S (not 80S) ribosomes, few introns, bacterial promoters, bacterial RNA polymerase, RNA not capped or poly-adenylated
• Chloroplasts evolved from free-living cyanobacteria
• Cyanophora paradoxa has a chloroplast-like endosymbiont ('cyanelle') resembling a bacterium
• Genes were transferred to nucleus during evolution (possibly via RNA), an ongoing process
• Possible reasons for gene transfer: higher mutation rate in chloroplast, Muller's ratchet (chloroplasts are asexual so accumulate mutations), better regulation of genes in nucleus
• Some genes (e.g. RNA polymerase) are nuclear in some plants but plastid-encoded in others
• Epifagus (beech drops) and Plasmodium (malaria) are like non-photosynthetic, parasitic plants
• Some protists contain 'hydrogenosomes' - like mitochondria with genome completely lost

Chloroplast protein import

• Transit peptide causes a protein to be imported into chloroplast, via TOC and TIC complexes (Translocase of Outer/Inner Envelope); transit peptide is then removed
• Some proteins have a second transit peptide for import across thylakoid membranes (at least four different thylakoid import mechanisms are known)
• Foreign proteins can be targeted to chloroplasts by artificially adding transit peptides

Mitochondria

• Mitochondria are surrounded by a double membrane
• Inner membrane is site of electron transfer chain in respiration; matrix is site of Kreb's cycle
• Yeast 'petite' mutants are slow growing due to defective mitochondrial gene, impairing respiration - mutation shows cytoplasmic inheritance (not normal inheritance patterns)
• Cytoplasmic male sterility in maize is a maternally-inherited mitochondrial mutation
• Mitochondrial genome is large and variable, with lots of non-coding and repeat sequences - encodes RNAs, ribosomal proteins, cytochromes, NADH dehydrogenase, etc
• Mitochondrial have bacteria-like 70S ribosomes (protein synthesis inhibited by antibiotics)
• Altered codon usage, caused by RNA editing
• Mitochondria use an altered genetic code (different in animal/plant/yeast mitochondria)
• There has been extensive and ongoing transfer of mitochondrial genes to nucleus during evolution (more than in plastids, because mitochondria entered cells earlier in history)
• Proteins targeted to mitochondria by pre-sequences are imported by TIM/TOM complexes
• Some sequences can target proteins to both chloroplasts and mitochondria (dual targeting)

Respiration

Aerobic respiration

• Respiratory quotient = CO₂ output/O₂ consumption   (indicates type of fuel being burned)
• Glycolysis - sucrose to pyruvate - net synthesis of 4 ATP and 4NADH per sucrose molecule
• Alternatively, sugar can be broken down by 'oxidative pentose phosphate pathway' (usually only at times of good supply), generating NADPH; intermediates can be used in biosynthesis (ribose for synthesising DNA/RNA, erythrose for synthesising phenolics)
• Kreb's cycle oxidises pyruvate to CO₂, producing 4 NADH, 1 FADH₂ and 1 ATP
• NAD-malic enzyme in plants can bypass part of Kreb's cycle, converting malate to pyruvate
• NADH fuels oxidative phosphorylation (electron transfer chain in mitochondrial inner membrane creates an H⁺ gradient that drives ATP synthesis)
• Energy charge (EC) = ([ATP]+ ½[ADP]) / ([ATP]+[ADP]+[AMP])
• High energy charge promotes biosynthesis and inhibits ATP generation
• Control of reactions in glycolysis:
  Phosphofructokinase activated by Pi; inhibited by ATP, citrate, PEP (a product of glycolysis);
  Pyruvate kinase activated by ADP; inhibited by ATP and citrate
• Alternative oxidase (AOX) bypasses last two steps of electron transfer chain, generating heat rather than ATP (uncoupling) - this is not sensitive to cyanide (which poisons cytochrome c)
• AOX is used by Voodoo lily in a 'respiratory burst' to release volatiles and attract pollinators
• AOX is activated by a high concentration of pyruvate, citrate and NADH

Anaerobic responses in plants

• Anaerobic respiration by microbes in soil reduces nutrient availability to plants (by reducing NO₃^- to N₂ and Fe³⁺ to Fe²⁺) and produces poisons (H₂S, acetic acid, butyric acid)
• Critical oxygen pressure (COP) = oxygen pressure at respiration is slowed
• COP depends on metabolic activity of cells, and is lower at low temperatures
• Pasteur effect - CO₂ production increases at very low O₂ levels, due to anaerobic respiration
• Energy requiring processes (e.g. ion uptake) are slowed in response to hypoxia
• Ethylene is produced by enzymes that are triggered by hypoxia
• Plants in wet environments have aerenchyma (hollow channels formed by programmed cell death, triggered by ethylene) that provide oxygen to roots
• Anaerobic proteins (ANPs), e.g. alcohol dehydrogenase, are produced in anaerobic conditions
• Anaerobic response is similar to heat shock (existing protein synthesis is shut down, new genes are transcribed and preferentially translated) but heat shock is faster and ubiquitous
• Anaerobic fermentation in plants regenerates NAD, allowing glycolysis to continue:
  pyruvate --> acetaldehyde + CO₂   (catalysed by pyruvate decarboxylase)
  acetaldehyde + NADH --> ethanol + NAD⁺   (catalysed by alcohol dehydrogenase)
• Plants have different alcohol dehydrogenase (ADH) genes
• ADH mutants can be screened for with allyl alcohol, which ADH converts to a toxin
• Pollen expresses ADH - aerobic fermentation, perhaps due to high energy requirements
• Sudden return of oxygen can lead to production of toxic reactive oxygen species (ROS), which are broken down into H₂O₂ by superoxide dismutase, then into H₂O + O₂ by catalase
• Net yield: 2 moles of ATP per mole of hexose in fermentation, 36 in aerobic respiration
• Plants have haemoglobins - for oxygen sensing? transport? storage?
• Engineering bacterial haemoglobin into plants improves growth and productivity

Peroxisomes

• Organelles containing oxidases (which generate H₂O₂) and catalase (which detoxifies H₂O₂)
• Involved in photorespiration and fatty acid b-oxidation
• b-oxidation - successive shortening of fatty acid chain, producing 2-carbon acetyl coA
• Glyoxylate cycle converts 2C acetyl coA to 4C organic acids (important in germinating seeds, pollen, senescence and starvation) - succinate is exported for gluconeogenesis
• Two models of peroxisome biogenesis - 'growth and division', and 'ER-vesiculation'
• Peroxisomal targeting sequences:
  PTS1 - widely conserved in evolution, not cleaved off, not always at protein terminus;
  PTS2 -  targeting sequence that is cleaved after import
• Some proteins imported by peroxisomal membrane proteins, but complexes can also be imported (molecules that don’t have targeting sequences can 'piggy-back' on those that do)
• ABC transporter imports acyl coA molecules (comatose mutant cannot mobilise storage lipid)
• Leaf peroxisomes can be converted into glyoxysomes in dark conditions
• Protein location in cells can be detected with immunogold labelling (gold bound to antibodies)

Cell expansion

• Growth = an irreversible increase in volume (cell division without expansion is not 'growth')
• Gamma-plantlets (seedlings irradiated with gamma rays) continue to grow normally for a while by cell expansion even though their cells cannot divide
• Cell division occurs in meristems
• Meristem cell is typically 10µm long; mature cell may be 1000µm long (mostly vacuole)
• Cell expansion is promoted by auxins, cytokinins and gibberellins
• Cell expansion is usually inhibited by abscisic acid and ethylene
• Low pH promotes cell expansion - auxin may promote growth by acidifying cells
• Cell expansion is driven by turgor pressure and resisted by the cell wall
• Lockhart equation:   rate of expansion, R = m(TP - Y) where TP is turgor pressure, m is extensibility, and Y is yield threshold (minimum turgor pressure for expansion to occur)
• Extensibilities of tissues differ - for example, in rhubarb stalks the interior is compressed but epidermis is under tension
• Turgor pressure can be measured by inserting tube from a micro pressure meter into cell

Composition of cell wall

• Thin primary cell wall is deposited while cells is growing; secondary cell wall deposited later
• Cell walls is ~30% cellulose - girder-like microfibrils held together by hydrogen bonding
• Matrix components of cell wall: hemicelluloses (~30%), pectins (~30%), others (~10%)
• Hemicelluloses: xylans (mainly in monocots), xyloglucans (mainly in dicots), mixed-linked glucans (only in expanding cell walls of grasses), mannans
• Pectins (e.g. galacturonans) - flexible, hydrophilic, jelly-like acidic polysaccharides - 'loosen' and 'lubricate cell' wall, introducing water and facilitating molecular movement
• Xyloglucan hydrogen-bonds with cellulose, forming 'tethers' that bind together microfibrils

Action of enzymes in cell wall

• Expression of cellulase (which can cut xyloglucan) is increased by auxins and ethylene
• Expression of pectinase in fruit is increased by ethylene (softens tissue during ripening)
• Xyloglucan endotransglycosylase (XET) cuts and re-forms xyloglucan tethers
• Donor substrate of XET is high-MW xyloglucan, acceptor is high or low-MW xyloglucan
• Measured activity of XET in tissues corresponds with their growth rate
• Gibberellins and brassinosteroids (growth promoters) increase XET activity
• Arabidopsis has 33 XET genes, similar in action but regulated differently
• Peroxidases can 'tighten' cell wall by linking together phenolic side chains of polymers
• Peroxidase activity is inhibited by gibberellins
• Evidence for enzyme activity in the cell wall - insoluble enzyme products (e.g. lignin) are present, degree of pectin esterification decreases with age, turnover of polymers such as callose and xyloglucan can be demonstrated by radioactive or fluorescent labelling, perforation plates develop in xylem and sieve plates in phloem
• Surprisingly, transgenic tomatoes lacking pectinase ripen normally
• 'Yieldins' promote expansion by lowering yield threshold (mechanism unknown)

Expansins

• Expansins - proteins that allow cell walls to stretch slowly ('creep') when under tension
• Expansins are denatured by boiling, Hg, Cu or proteases, but not by methanol
• Optimum expansin activity is at pH 4 (acid growth)
• Adding protein extract restores creep to denatured cell walls (and bits of paper!)
• Expansins work by weakening hydrogen bonds involving cellulose
• Expansins occur in rapidly-growing plant tissues and in ripening fruit
• Beads impregnated with expansin trigger formation of leaf primordia at apical meristems
• Two classes: a-expansins work best in dicot cell walls, b-expansins best in monocots
• Arabidopsis has 30 different a-expansin genes

Plant genomics

Structure of plant genomes

• Arabidopsis genome: five chromosomes, 125 megabases in total (quite small), ~25,500 genes
• Chromosomes have of centromere, euchromatic (active) arms, and telomeres (at ends)
• Centromere is site of spindle attachment at meiosis - it contains heterochromatin (densely-packed, highly-methylated DNA with little transcriptional activity)
• Repetitive DNA: telomeric and sub-telomeric repeats, centromeric repeats, "heterochromatic knobs" (remains of old centromeres?), transposable elements, tandem gene repeats
• Type I transposons (retrotransposons) replicate through an RNA intermediate
• Type II transposons ("jumping genes") replicate through a DNA form
• Link between high transposon numbers and heterochromatin - do transposons target 'quiet' regions? Are they preferentially lost from active ones? Do they render DNA inactive?
• Autonomous transposons encode proteins required for movement (e.g. transposases)
• Expressed Sequence Tags (ESTs) - mRNAs reverse-transcribed back into DNA - are used to identify expressed genes (but are hard to generate for very small peptides)
• Genes also identified by: similarity to existing genes, open reading frames (no stop codon), base pair composition, codon usage, elements of gene structure (splice sites, promoters, etc)

Plant gene expression

• Genes expressed in response to: environmental cues, spatial cues, developmental cues, and growth substances (auxins, gibberellins, etc)
• Three nuclear RNA polymerases: polymerase I (large ribosomal RNAs), polymerase II (protein-coding RNAs, snRNAs), polymerase II (5sRNA, tRNAs - small and stable)
• Gene chips - immobilised EST fragments hybridised with RNA populations to reveal which genes are being expressed (disadvantage - shows transcription but not proteins themselves)
• Cis-transcriptional regulation by promoter elements - proximal elements (TATA box) bind RNA polymerase II; distal elements (usually composed of transcription factor binding sites) responsible for gene-specific expression patterns
• Enhancers - orientation-independent distal promoter elements that stimulate gene expression
• DNA methylation and histone methylation/acetylation have transcription-silencing effect
• Epigenetic mutations (e.g. loss of red pigment in maize) caused by DNA methylation
• Post-transcriptional regulation by RNA binding proteins (in circadian rhythms, floral patterning), alternative RNA splicing (FCA - full protein not expressed early, preventing premature flowering) and by small RNAs
• 'Dicer' (double stranded ribonuclease) cuts double-stranded RNA - viral defence mechanism
• sRNAs incorporated into RIS (RNA-induced silencing) complex, which binds single-stranded homologous RNA - RIS mediates synthesis of complementary RNA strand, which is chopped up by dicer

Plant genome dynamics

• Comparing Arabidopsis ecotype Columbia to other ecotypes (e.g. Landsberg erecta), 25,274 SNPs (single nucleotide polymorphisms) detected over 82Mb (1 per 3.3Kb)
• Insertions/deletions ("indels") in exons largely multiples of 3 base pairs (not frame-shifting)
• Variation caused by unfaithful DNA replication (including 'slippage' in repeated regions) and spontaneous lesions due to DNA damage (e.g. depurination, deamination) - most mistakes are corrected (proofreading/mismatch identification)
• Recombination at meiosis - chromosomes pair and exchange DNA at chiasmata
• Unequal crossing over between duplicated genes can lead to tandem arrays
• Transposons cause genetic change - chromosomal breakage, transposition of genes to new positions, insertion within genes causing loss of function, deletions (a gene in between two transposons may be accidentally excised during transposition or chromosomal recombination)
• Transposon replication involves recognition of terminal inverted repeats by transposases
• Polyploidy - duplication of entire genome - is rare in animals but common in plants
• Autopolyploidy (duplication of one genome via non-reduced gametes) disrupts meiotic pairing and can cause sterility - this instability is reduced  by diploidisation
• Wheat - three genomes - divalent pairing of homologous chromosomes at meiosis despite intergenomic homology, controlled by locus Ph1 on chromosome 5B ('nullisomics' lacking this chromosome show considerable multivalent pairing)
• Allopolyploidy (merging of two different genomes) fundamental in speciation
• Mutagens:
  ethyl-methane sulphonate (EMS) - adds ethyl group, causing mispairing of G with T;
  intercalating agents (e.g. acridine orange) - mimic pairs and cause single insertions/deletions;
  UV light and aflatoxin b - damage nucleotides and prevent specific pairing;
  ionising radiation (x-rays, gamma rays, fast neutrons) - produces ions in tissue and causes deletions

Gene mapping

• Positions of genes on chromosomes can be deduced by frequency of recombination
• One genetic map unit (centimorgan) is distance between marker pairs for which one product of meiosis out of 100 is recombinant (recombination frequency, r.f. = 0.01)
• For unlinked genes (on different chromosomes), recombination frequency = 0.5
• Recombination frequency will be underestimated due to double recombination
• Molecular markers used in genetic mapping:
  Restriction Fragment Length Polymorphisms (RFLPs) - length cut out by restriction enzymes;
  Simple Sequence Length Polymorphisms (SSLPs) - repetitive sequences prone to slippage;
  Cleaved Amplified Polymorphic Sequences (CAPS) - SNPs creating restriction enzyme sites;
  Randomly Amplified Polymorphic DNAs (RAPDs) and Amplified Fragment Length Polymorphisms (AFLPs) - require no sequence information - used in divergent populations

Manipulation of plant genomes

• Protoplasts may take up DNA 'naturally' (aided by polyethylene glycol or electric pulses)
• 'Biolistic' approach - fire DNA-coated gold particles into cells - useful for transient transformation (where regeneration is not required)
• Using Agrobacterium tumefaciens (which normally infects wounds and causes galls) as a vector
• Agrobacterium contains a Ti plasmid, which incorporates its genes into genome of host cells
• vir genes encode machinery for transferring the T-DNA region of plasmid into host cells
• Normal Agrobacterium T-DNA causes synthesis of auxin and cytokinin-like molecules promoting gall growth (oncogenic) and opines (substances used by bacterium)
• T-DNA - bounded by borders recognised by vir genes - doesn't have to be on same plasmid
• In genetic manipulation, T-DNA is modified to remove oncogenes, insert Multiple Cloning Sites (for insertion of new constructs), and add selectable marker (e.g. for antibiotic resistance)
• First generation transformants may be chimaeras of transformed and untransformed cells - breed and perform selection on second-generation plants to obtain true transformants
• Dipping Arabidopsis flowers in Agrobacterium transforms ovule primordia, producing entire plants derived from transgenic egg cells (no chimaeras), often with multiple T-DNA insertions

Transgenic techniques as genomic tools

• T-DNA insertions: in coding regions (loss of gene function), in promoters (disruption of gene expression), in introns (gene disruption or no effect)
• Important to check linkage of a transgenic phenotype with inserted T-DNA
• T-DNA flanking sequences can be identified easily by TAIL-PCR
• T-DNA can be used to transfer transposons between species - to ensure stability, put transposase in separate insertion, breed plants, and select those that lose transposase
• Activation tagging - insertion of T-DNA containing viral enhancers that cause massive overexpression of certain genes - often observed even if gene is member of multi-gene family
• Antisense (RNA interference) - suppression of gene expression using complementary RNA, which interacts with mRNAs to produce double-stranded RNA that is chopped up by dicer
• Overexpressing genes - use constitutive promoter to express gene everywhere, or a promoter from another characterised gene to express gene in a specific place
• Markers for identifying gene expression patterns:
  GUS (beta-glucuronidase) from E. coli - used in chemical assays on dead tissue, very stable;
  GFP (green fluorescent protein) from jellyfish - easy to use but not quantifiable
  Luciferase from fireflies - catalyses bioluminescence when luciferin is added

Chloroplast transformation

• Genes inserted into chloroplasts by bombardment with small tungsten particles
• Useful for expression of prokaryotic or modified chloroplast genes
• Advantage - amplification (many chloroplasts per cell)
• Disadvantage - expressed in every tissue where photosynthesis occurs

Plant biotechnology

• Might be possible to increase yield by decreasing photorespiration:
  introduce C₄ metabolism (fixation of CO₂ in cytoplasm by PEP carboxylase) into C₃ plants?
  manipulate key enzymes such as RuBisCO?
• Alternatively, change balance of starch metabolism to 'drive' photosynthesis:
  e.g. FDA (fructose 1,6-bisphosphate aldolase) converts fructose 1,6-bisphosphate into triose phosphate leading to starch production - starch formation can be increased by inserting E. coli version of gene, not sensitive to triose phosphate inhibition, into chloroplast
• Inserting Bt (Bacillus thuringiensis) toxin gene into chloroplasts provides pest resistance
• Hypersensitive response to fungal attack - place avr gene for which plant has corresponding vir gene under control of promoter sensitive to a wide range of pathogens - makes a specific response more general
• Expressing tobacco mosaic virus coat protein gives resistance - due to RNA-induced silencing
• Glyphosate-resistant plants ("Roundup Ready") - glyphosate normally inhibits EPSPS (amino acid synthesising enzyme) - insertion of mutant bacterial EPSPS gives herbicide resistance
• More slowly-ripening tomatoes - antisense inhibition of cell wall-degrading polygalacturonase
• High-lysine soybeans - inserting E. coli enzymes not sensitive to end-product inhibition
• Golden rice - three enzymes (entire pathway) inserted to allow vitamin A synthesis
• Phytoremediation - using genetically modified plants to remove pollutants from environment
• 'Pharming' - production of pharmaceuticals and other products in genetically modified plants
• Risks of biotechnology - health risks due to allergenicity or production of toxic by-products, spread of antibiotic resistance genes, spread of transgenes to weeds, impact on Third World?
• Another potential problem - transgene silencing (high levels of RNA invite attack by dicer)

Plant-microbe interactions

Plant pathogens

• May be opportunistic (many bacteria), or may be obligate pathogens (viruses, many fungi)
• Biotrophs employ stealth, necrotrophs overcome a plant by brute force
• Hemibiotrophs start as biotrophs and later become necrotrophic
• Some necrotrophs deliberately induce apoptosis then feed on dead cells
• Pathogens enter plants via stomata or via wounds
• Examples of plant pathogens:
  Downy mildew - an obligate biotroph, suppressing cell death and other plant responses;
  Pseudomonas syringae - hemibiotrophic bacteria, initially attacking living plant cells;
  Erwinia carotovora - necrotrophic, secreting enzymes that kill plant tissue;
  Pseudomonas aeruginosa - infects both plants and animals!

Pathogen resistance in plants

• Plants have preformed defences (e.g. cuticle wax) and pathogen-induced defences
• Hypersensitive response (hrp) genes involved in pathogen resistance
• Some hrp genes are highly-conserved - similar to pathogen resistance genes found in animals
• avr (avirulence) genes in pathogens are recognised by vir genes in plants, triggering resistance
• Absence of either the avr gene or the vir gene leads to susceptibility

Symbiotic associations with rhizobia

• Rhizobia - common, Gram-negative soil bacteria, which reduce N₂ to ammonia
• Related to Agrobacterium, but lack a Ti plasmid
• Rhizobia occur in root nodules of legumes (a very successful, diverse plant group)
• Different types: Rhizobium, Bradyrhizobium (slow-growing), Azorhizobium (free-living)
• Plants secrete flavonoids (phenolic compounds) that attract rhizobia (chemotaxis)
• Host-specific attractants attract specific Rhizobium species
• Rhizobia contain nod genes that are only expressed in contact with host plants - specific flavonoids activate nod genes of specific rhizobia, and inhibit those of incompatible species
• nodD is a 'global activator' that activates all other nod genes - binds flavonoids?
• All Rhizobium species contain nodA, nodB, and nodC genes, plus some species-specific ones
• nod genes work together to synthesise a 'nod factor' that triggers root nodule formation
• Signal triggers root hair deformation ("had" activity) and hair curling ("hac" activity)
• Rhizobia are trapped in curled hair - they can produce hydrolytic enzymes to degrade plant cell walls and facilitate entry
• Infection thread forms - invagination of plant cell membrane traps rhizobia
• Indeterminate nodules (with distinct zones) form from inner cortex of root
• Determinate nodules (with cells all at a similar developmental stage) form from outer cortex
• Infected cell forms phragmoplast - rhizobia inside are surrounded by peribacteroid membrane
• Rhizobium signals alter auxin/cytokinin balance, activating cell division and nodule growth
• Nodule number tightly regulated - too many would result in parasitism, not symbiosis
• ENOD genes produce peptide hormones (unusual in plants) that regulate nodule formation

More notes and essays

© Andrew Gray, 2004