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 (Vmax = 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 + Pi
- 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 Pi
- 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 + Pi
- 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
Pi), 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 + Pi (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
- Pi (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; Pi 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
+ Pi
- 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 (yw) = solute potential (ys) + hydrostatic potential (yp) + gravity potential (yg)
- 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
- Vmax is the reaction velocity of an enzyme at
infinitely high substrate concentration
- Km (Michaelis-Menten) constant is the substrate
concentration at which velocity is half Vmax
- Michaelis-Menten equation for an idealised enzyme: V = (Vmax [s]) / (Km
+ [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(Cin/Cout)] 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, Cin and Cout are concentrations of ion on
either side of membrane
- For a univalent cation at 25°C, this simplifies to: DE = 59 log(Cout/Cin)
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