Grand Green Flashcards
Global population
10.4 billion in 2080?
Famine
1 billion do not have enough food
hunger and malnutrition
9 million people die each year (more than AIDS, malaria and tuberculosis combined); the silent pandemic; 6.3M COVID deaths ‘20-‘22
Food cost fluctuations
- weather
Non-food uses of plants
- feed
- biofuel
- fibre
- construction
land use for agriculture
50%
agricultural land use for meat
77%
crops provide
- 82% calories
- 63% protein
additional challenges
- less land
- less predictable water
- less fertiliser
- fewer pesticides
8 main crops of the world
- maize
- wheat
- rice
- barley
- cassava
- oilseed rape
- sugarcane
- soy
most cropland is used for
- cereals
- coarse grains
- oilcrops
crop domestication
- no fruit abscission
- more and bigger fruits
- loss of daylength dependence
- determinate growth
- colour variation
- loss of vernalisation requirement
- increased seed number
- reduced seed shattering
- reduced height
- reduced dormancy
conventional backcrossing
- visual selection of F1 plants that most closely resemble recurrent parent
marker-assisted backcrossing
using background markers that allow selection of F1 plants with the most recurrent parent marker genes, and the smallest percentage of donor genome
speed breeding
- providing optimal environmental conditions that enable faster growth and reproduction
- light, temperature, photoperiod, and humidity
How to increase genetic variation?
- increasing germplasm (create seed stock centres)
- mutagenesis
- transformation
- genome editing
how do you replace bases with CRISPR-Cas9
inefficient HR
virus induced gene editing (VIGE)
CRISPR-Cas9 transgenic plant with virally transmitted sgrna
Precision Breeding Act: 23rd March, 2023
genetic changes which could have arisen through traditional breeding or natural processes
key challenges of the future
- drought-resistant
- flood-tolerant
- disease-resistant
- low N; nodulation
- low P; AM symbiosis
- improved photosynthesis
- flowering time
- better morphology
additional challenges
- germination
- cold/frost damage
- biofortification
- morphology
- new crops
- weed control
stress
Suboptimal environmental condition that adversely affects the growth and development of a plant
abiotic stressors
- temperature
- water
- CO2
- light
- nutrient
- salinity
- heavy metal/xenobiotic poisoning
- mechanical
biotic stressors
- pathogens
- pests
- plants
stress reduced crop yields by
more than 50%
stress responses
- tolerance
- avoidance
phases of stress
- alarm
- acclimation
- maintenance
- exhaustion/recovery
chronology of a stress response
- immediate responses
- gene expression and metabolism
- physiology
- growth and development
ROS
can damage proteins, lipids, DNA, causing cellular dysfunction
long-distance signals in plants
- ROS/calcium waves
- electric currents
- hydraulic signals
- pH
- eATP
- phytohormones
- hormone-like peptides
- miRNAs
water regime terminology
- water logging
- partial flooding
- complete flooding
flooding is a major
financial threat (US crop production 2013-2017)
intensity, timing and duration
of floods are changing
Why is submergence a threat to plants?
- water turbidity results in carbon starvation and slow diffusion of oxygen
- fermentation
- soil redox potential decreases
- reduced photosynthesis
one way to look at protein expression levels
relative across time/place
ways of studying interactions
- mass spectrometry
- biomolecular fluorescence complementation assay
flooding assays
- hypoxia tolerance assays in vitro
- submergence assays in vivo
natural strategies to withstand flooding stress
- germination
- roots
- stems
- leaves
Specific flooding stress adaptations
- anaerobic germination
- root repatterining
- hydraulic repatterning
- aerenchyma
- heterophylly
- LGFs
GWAS
- association of a specific trait with SNPs across hundreds of accessions reveals interesting regions
enzyme-engineering approaches
- structure-guided rational design (rational mutagenesis)
- deep mutational scanning
- synthetic biology
- de novo protein design
- random mutagenesis
- directed evolution
drought
ranks as the single most common cause of severe food shortages in developing countries
drought strategies
- desiccation
- avoidance
- tolerance
0.2% of the angiosperms are
dessication tolerant
short-distance water movement
- diffusion and osmosis
long-distance water movement
pressure bulk flow
water path
- from root to soil
- thru plant
- from leaves to atmosphere
factors affecting water movement
- xylem anatomy
- leaf vein pattern
- guard cell density and opening
- plasmodesmata
- aquaporins
- soil properties
- root architecture
- cell wall permeability
ROS formation
- Membrane damage
- Protein aggregation
- Impairment of photosynthesis
and other cellular functions
why is drought a stress?
- low energy stress
- low oxygen stress
- osmotic stress
short-term water responses
- balance
- protection
water balance
- stomata
- osmolyte
- aquaporin
protection
- LEA
- chaperones
- AOs
long term flood responses
- source and sink relations
- drought escape
source and sink relations
- roots
- hydraulics
- epidermal wax
- leaf abscission
- reduced stomata
- reduced shoot growth
drought escape
reproductive phase transition
monitor drought
- soil monitoring
- plant monitoring
soil monitoring
- humidity
- drip irrigation
- partial root zone drying
plant monitoring
- thermal imaging
biological innovations for drought
Osmotic adjustment
Regulation of stomata conductance Improved water use efficiency Improved photosynthetic rate Delayed senescence
Root architecture
Regulation of reproductive phase transition
ways of improving drought
- chemicals
- memory
- drought-tolerance
drought chemicals
- osmoprotectants
- growth effectors
drought memory
seed priming
forward genetics
- phenotype to gene
1) mutagenesis
2) screen
3) map [GWAS]
4) complementation testing
5) KOs
6) expression
backwards genetic
- gene to phenotype
manipulating relevant genes
- transgenic approaches
- breeding (MAS)
- gene editing
conventional breeding
Search for progeny combining both parental traits
VERY LONG AND TEDIOUS PROCESS (8-12 selection generations)
BASED ONLY ON PHENOTYPE
GENOTYPE-BASED selection of progeny
- Progeny with “tolerance” trait identified
- Progeny with highest % of high-yield parent identified
Gene editing
IF YOU KNOW WHAT IS THE CAUSAL GENE/MUTATION
HIGHLY PRECISE, FLEXIBLE, AND FAST GENOME MANIPULATION
3400 calories
per person per day
Average increase
~15 extra daily calories
per year; (0.6% increase per year)
78 million additional loaves of bread every day next year
= 15 million additional tons of wheat next year
= 51,000 additional km2 of wheat next year
Or
= 2,340,000 additional km2 of cows next year
Average 1.1% yield improvement
per year since 1960
Average 0.3% increase in land use
per year
Cut down ~15 billion trees
a year
~ 30% of humanities
CO2 emissions
you can measure the evolution of a
protein site
India crashed into mainland Asia
- Himalayas began to erode
- Weathering of Himalayas caused to CO2 to plummet
What are the consequences of photorespiration?
No carbon gain
No growth
Risk loosing both N and C
30-50% of all plant energy spent undoing this reaction at current atmospheric CO2.
If evolution cant change rubisco perhaps it can change its environment!
Ratio O2 : CO2
Atmosphere 500 : 1
Solution 10 : 1
Most plants
Photosynthesis in one cell
C4 plants
Photosynthesis distributed across two cells
C4 photosynthetic partitioning evolved independently
> 70 times
Independent origins plus different ages
- = huge diversity in anatomy
- huge diversity in biochemistry
light
- not equally absorbed by the earth
- scattered by the atmosphere
- only a fraction is used for photosynthesis
- light environment is constantly fluctuating
- position in canopy has large effect on light levels
- chlorophyll light interception responses
Three ways people have tried to improve responses to light availability
Speed up rubisco reaction time.
Speed up NPQ reaction time.
Change light absorption in canopy.
if you have improved a characteristic what should you ultimately measure?
- yield
Chloroplast descended from photosynthetic bacteria that lived in
upper levels
Photosynthesis using infra red light emitted from
deep sea hydrothermal vents
Functional reconstitution
in vitro
Rhizosphere zone
immediately around roots
Endosphere inside roots but extracellular
(nb phyllosphere (leaf), spermosphere (seed))
5-20% of plant photosynthate is
exuded by roots
Microbiome detection
Metagenomics (DNA), metatranscriptomics (RNA), metaproteomics
Microbiome structure
Operational taxonomic Unit (OTU) i.e. bin sequences to >= 97% identity, now amplicon sequence variants (ASV) most common
Bray Curtis dissimilarity
- difference or dissimilarity between two samples based on their species composition or abundance in ecological studies, particularly when analyzing community structure
- value between 0 and 1, where 0 indicates identical communities and 1 indicates completely different communities
Microbiome mapping
- Fluorescently labelled bacteria can be counted by flow cytometry
- Bacteria can be tracked in microfluidic devices
- Global mutagenesis (TnSeq, BarSeq) used to determine effects of mutation
All nodulating plants are in
Eurosid 1 clade
Fabales,
95% in Legume Family = Fabaceae (or Leguminosae)
3rd largest family of plants
19,400 species
Three subfamilies of Fabacea
1:Mimosoideae
2:Caesalpinioideae 3:Faboideae (Papilionoideae)
Not all nodulate,
rare in Caesalpinioideae
Parasponia
only non-legume host (Rosales)
Soybeans
twice as much protein per acre as any other major vegetable or grain crop, 5 to 10 times more protein per acre than land for grazing animals to make milk, and up to 15 times more protein per acre than land set aside for meat production.
Medicago truncatula
model indeterminate plant (375 Mb)
Lotus japonicus
model determinate plant (470 Mb)
Both M. truncatula and L. japonicus have
small diploid genomes, easy to transform
Glycine max (soybean)
1.1.Gb polyploid genome
Synteny mapping
analysis and comparison of the relative order of genes or genetic markers between different species or within different regions of the same genome. It helps identify regions of conserved gene order across different species, which can provide insights into evolutionary relationships and functional genomics.
Most rhizobia are Five main groups of alpha-proteobacteria
i) Rhizobium; ii) Bradyrhizobium; iii) Azorhizobium; iv) Sinorhizobium; (renamed Ensifer) v)
Mesorhizobium
Beta proteobacteria
(e.g. Burkholderia and Cupriavidus)
Phosphorus (P)
The 11th most abundant element in the earth’s crust
The 5th most abundant element in a plant
The 2nd most common limiting macronutrient for plant growth
Phosphorus uses
- Energy donors ATP, ADP, AMP
- Phospholipids
- Nucleic acids - DNA, RNA
- Starch/sucrose synthesis
- Protein modification, regulation of metabolic pathways such as energy transfer, and amino acid synthesis
Phosphate Depletion
- Stunted growth, chlorosis, increased levels of anthocyanins
- Increased root-to-shoot ratio
- Low metabolism & reduction in
photosynthesis - Sulpholipids/glycolipids replace phospholipids in membranes
- Delayed flowering and poor seed quality/low crop yields
- Reduced Nitrogen uptake
- Leaf drop
- Poor frost resistance
P is essential for
growth, functioning and reproduction of all life on earth
Pi fertilisers
boost plant growth and crop yields
Approaching a
period of “peak
phosphorus” as
depleted
deposits
Phosphate rock is a
non-renewable resource
90% of the world’s Pi rock reserves are in only
5 countries
~80% soil-applied phosphate remains
unavailable to the plant (Hinsinger et al., 2011)
P deficits cover
29% of the global cropland area impacting crop yields
71% of the cropland area had overall Phosphorus
surpluses
High P fertilizer application relative to crop P use (Low Phosphate Use Efficiency) resulted in a greater
proportion of the intense P surpluses
(>13 kg of P·ha−1·y−1) globally
PSR in shoots (systemic signals delivered through xylem and phloem translocation system):
- Increased Pi recycling and mobilization
- Reduced rate of photosynthesis
- Increase in sugar concentration
- Increase in anthocyanin accumulate
- Lipid remodelling
- Shoot growth retardation
PSR in roots (local):
- Increase in Pi uptake, transport and translocation
- Changes in Root System Architecture (RSA)
- Increased secretion of Organic Acids and Acid Phosphatases * Changes in metabolic pathways
- Lipid remodelling
- Exudation of flavonoids and strigolactone (SL)
- Interaction with Arbuscular Mycorrhizal Fungi
necessary?
attenuated
RT-PCR
showing homolog transcript levels
Spatial expression pattern
- are different cells doing the same thing?
Concanamycin A:
Potent inhibitor of V-ATPase resulting in vacuolar alkalinisation and fragmentation
iTRAQ analysis
quantitative proteomics technique used to measure the relative abundance of proteins across multiple samples in a single experiment. It allows researchers to compare protein expression levels between different conditions (e.g., different tissues, time points, or experimental treatments) in a highly efficient and reproducible manner
if you’re testing a protein you could test its
homology
if you’re doing a mutant analysis you could do
- mutant combos
- OX lines
Reciprocal micrografting
- ## By swapping the root and shoot tissues between two plants, researchers can determine whether certain traits or characteristics are regulated by the root or the shoot.
When would you use YFP?
- enhanced photo stability for live cell imaging
- efficient at folding v fast
When would you use mCherry?
- better penetration
- higher photo stability
plant symbiosis
Plants can derive up to 100% of their phosphorus needs and around 40% of their nitrogen needs from AM fungi
what percentage of plants are non-mycorrhizal?
~7%
what percentage of plants have AMS?
72%
AMF
570MYA
AMS enhances overall maize grain yield by
~30%
Top-5 crop diseases are all filamentous!
- Stem rust
- Rice blast
- Late blight
- Soybean rust
- Corn smut
Top-5 tree diseases are all filamentous
- Dutch elm disease
- Chestnut blight
- Sudden larch death
- Ash Dieback
- Blue stain disease
Three main crop protection strategies
- Resistance breeding
- Agrochemicals (fungicides)
- Good sanitation
Wheat rust
- 7,500 BC
- Aristotle’s time (384-322 BC)
- ‘Robigalia’ festival in Rome on April 25: Sacrifice puppy to Robigus to avoid rust
- Problematic in 17th century Europe
Norman Borlaug
Developed semi-dwarf, high-yield, disease-resistant wheat varieties.
Uganda, 1999
Devastating rust epidemic
New stain broke through existing R genes
Spreads by wind.
strain UK-01
- 2013: first rust in UK in 58 years
- 20% of UK wheat varieties are resistant to UK-01
Cladosporium fulvum
- serious losses in greenhouse-grown tomato
- new outbreaks in UK every year since 2000
Edman sequencing
PCR with degenerate primers
- uses primers containing mixed or ambiguous bases to amplify DNA sequences with some variability
- useful when the exact target sequence is unknown
- Gene discovery – Amplifying homologous genes from different species.
- Metagenomics – Detecting diverse microbial genes in environmental samples.
- Evolutionary studies – Identifying conserved gene regions across species.
- Pathogen detection – Detecting viral or bacterial genes with some sequence variation
Map-Based Cloning
- Genetic Mapping with Molecular Markers
- Fine Mapping
- Physical Mapping & Candidate Gene Identification
- Gene Validation
Gene-for-gene transient assays
- Selection of Genes
- Cloning & Expression (CaMV 35S)
- Agrobacterium-mediated transient expression (Agroinfiltration) or viral vectors to deliver genes into plant cells
- Co-Infiltration into Host Plant (Control treatments include empty vector or non-interacting genes)
- Observation of Hypersensitive Response (HR); visually assessed within 24–72 hours as necrosis or electrolyte leakage (measured with conductivity assays).
Tomato brown rugose fruit virus
- First identified in Israel (2014) – now spreading worldwide
- Outbreaks in the UK (one in 2019, four in 2020)
Eupatorium yellow- vein virus (EpYVV)
autumnal appearance of eupatorium plants in summer
Tobacco mosaic virus
- Work in the 1880s demonstrated that the tobacco mosaic disease was caused by an infectious agent (Mayer, 1886) that could pass through porcelain filters (Iwanowski, 1892)
- Beijerinck (1898) realized the agent was unlike any previously identified pathogen and coined the term VIRUS
- 1930s Stanley crystalized TMV…. Viruses are a very distinct form of life
Virus-infected plants show greater resistance to
drought
2020
Approx. 2000+ viruses and satellites
virus couples
Some viral diseases are now recognised as being caused by two or more viruses – with a single virus not causing disease symptoms…
Papaya “sticky” disease
PMeV infects with a second virus such as PMeV
Most vectoring of plant viruses is via
invertebrates, especially aphids (66%)
The “number 1” model plant pathogen
Pseudomonas syringae*
Phytoplasmas are
obligate parasites with small genomes (<1Mb) and depend on host cells for some biosynthetic processes
Plant-feeding insects damage plants and
act as vectors for plant diseases
psammophory
Over 200 species of plants coat themselves in sand, which makes them less appealing to herbivores
biological communication
“Information transmission that is fashioned and/or maintained by natural selection”
Signals are
traits whose value to the signaler is that they convey information to receivers
Communication
if signaling plant derives a fitness benefit from conveying information to others
semiochemical
a generic term used for a chemical substance or mixture that carries a message
Hormone
semiochemical produced in one part of an organism that exerts an effect in another part of an organism
Allomone
semiochemical produced and released by a living organism that benefits the donor (signal producer)
Kairomone
semiochemical produced and released by a living organism that benefits the receiver
Synomone
semiochemical that is adaptively advantageous to both the emitting and the receiving organism
Volatile organic compounds (VOCs)
- key agents of chemical communication for plants
- compounds that have a high vapor pressure and low water solubility
- Up to 36% of assimilated carbon may be released by plants as complex bouquets of VOCs
VOCs act in conjunction with other signals such as flowers or temperature to
communicate messages to pollinators
odour can be particularly important for
nocturnal animals
Could enhancing scent production in commercially bred flowers enhance both
appeal and pest/disease resistance?
Kin recognition in
competitive root interactions
Antagonistic root growth
- competition
- inhibition (allelopathy)
Studying VOCS
Plants producing essential oils are distilled and the extracted oils analyzed by gas chromatography, often coupled to mass spectrometry
Plant headspace is sampled using sorbents
- sampling of airborne organic pollutants
- ## Tenax TA, Chromosorb 106, Porapak N, and Carbopack F
Ways to measure volatiles
- sorbents
- Solid phase microextraction
- eNose
- Insect electroantennogram
Electronic nose (eNose)
Metal Oxide Sensors: when volatile compounds are adsorbed onto the surface of the semiconductors this generates a change in the electrical resistance which varies with the type of volatile compound and its concentration.
Challenge of testing hypotheses about volatile (airborne) signaling…
- Use of realistic (natural) volatile concentrations
- Use of single compounds rather than mixtures
- Enclosed plants – altered gas exchange (CO2, H2O, O2)
- Use of detached leaves (more sensitive to VOCs)
- Well-controlled, biologically meaningful stimuli (e.g. chewing damage involves mechanical and chemical stimuli such as saliva)
Scent production by snapdragons decreases after
fertilisation
Traps based on
plant volatiles and sex pheromones
Vernalisation is
the process by which prolonged exposure to cold temperatures promotes flowering.
Ageing enhances
flowering competence
Reproductive phase transitions are regulated by
microRNAs
Flowering time is a
domestication trait
P. syringae
- bacterial speck disease
- many strains
- also infects model plants
Plant domestication traits
- increased seed retention (non-shattering)
- increased size
- changes in shoot branching and stature
- loss of seed dormancy
- synchronous germination
- taste change: loss of bitterness, enhanced sweetness
- fruit colour polymorphisms
- change of flowering time
Bacterial growth assay (colony counts)
1) syringe infiltration by hand
2) make leaf extract; dilution series
3) count colony-forming units (CFU)
Phytophthora infestans
- single most important disease facing the potato industry
- €6 billion losses globally: control and damage
- also infects tomato
The history of potato blight
- Potato domesticated 7-10 kya in Peru/Bolivia, driving (pre-)Inca empire
- late blight until 1840s (co-evolved in Mexico with wild potato)
- Great Famine in Ireland was caused by strain HERB-1
- ~ 1M people died, ~1-2M emigrated
Virus-induced Gene Silencing (VIGS)
- RNAi technique to transiently deplete mRNA levels of specific genes in plants
- Tobacco Rattle Virus (TRV) RNA2 is transcribed from T-DNA of binary agroinfiltration vector
Cloned by association genetics
identifying and isolating a gene based on its statistical association with a particular trait in a genetically diverse population. This approach is often used in natural populations or breeding lines rather than controlled genetic crosses.
2009 milestone:
genome of P. infestans sequenced by large consortium
effectoromics
1) Sequence your pathogen
2) Select core effector candidates
3) Clone into binary PVX vector
4) Agroinfect wild relatives
5) Identify NLRs responsible for recognition by co-expressing wild-relative NLR candidates in N. benthamiana (by agroinfiltration)
6) Transform NLR into potato to introduce resistance against P. infestans
7) Stack NLRs into potato for durable resistance
GMO landscape remains challenging
BASF’s Fortuna (GM potato containing Rpi-blb1 & Rpi- blb2 from S. bulbocastanum) stopped in 2013 because of ‘uncertainty in the regulatory environment and threats of field destructions’.
