Week 4 Flashcards

Question

Why might protoplast transfection be used?

Answer 1

The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system

Answer 2

Gene-editing by expression of developmental regulators and de novo meristem induction in plants. RNA viruses and mobile guide RNAs for heritable plant gene-editing. Nanoparticles for delivering biomolecules to facilitate plant genome engineering

Answer 3

Immature embryos (cereals) Mature embryos Suspension cultures Protoplasts Hypocotyl explants Cotyledonary petioles (Brassicas)

Answer 4

Explants Regeneration Transgenic plants on selection medium

Answer 5

Remove existing meristem Introduce reagents Meristem formation Growth with fixed modifications Segregation of modification in offspring

Answer 6

Only really possible in tomaotes as other plants may struggle Also ineffcient

Answer 7

Single walled carbon nanotubes (CNTs) (~1 - 1000 nm) and carbon dots (~3 nm) Delivery using syringe through stomata DNA-CNTs traverse the cell wall and membranes and modifies DNA

Answer 8

T-DNA integration Regeneration (Somatic embryogenesis/ organogenesis)

Answer 9

Type, concentration and ratio of: Cytokines Gibberellin Auxin Other

Answer 10

Type Age Size Orientation

Answer 11

Mineral nutrients Vitamins Carbon sources pH

Answer 12

Temperature Light

Answer 13

Identify the spike with right stage of immature embryos (IEs) and isolate Centrifuge and co-cultivate IEs with Agrobacterium for 3 days Excise embryonic axis 5 days later and culture in resting medium and under hygromycin selection +5 weeks Culture in regeneration and rooting media 2 weeks + further 2 weeks Transfer to soil Analysis

Answer 14

Target tissue: Immature embryos Delivery method: Agrobacterium strain AGL1 Selection system: Hygromycin resistance alternative selection G148 Genotype: Fielder (US spring wheat) Constructs: initially pBRACT series, now superseded by pGGG

Answer 15

Transformation efficiencies of up to 25% Spring wheat cv ‘Fielder’ (Calculated 1 plant from 1 embryo)

Answer 16

Genes: PGA37/MYB118, Wus2, Bbm, Shoot Meristemless (STM) and Wox5 Meristematic and embryogenic genes

Answer 17

Continued expression of the genes can cause developmental defects. Plant regeneration using these technologies requires controlled expression or removal of the expression cassettes. So far, limited activity in dicots

Answer 18

GRF-GIF --> Growth regulation factor Significantly increased the transformation efficiency Reduced the time of transformation process (from 90 to 50 days) Reduced constrains that limit transformation efficiency (plant condition, embryo size …). Expands the range of transformable genotype. The GRF4–GIF1 technology results in fertile and normal transgenic plants

Answer 19

Expanded the transformable genotypes Allowed us to transform elite commercial varieties Varieties such as Skyfall, Reedling, Paragon and Cadenza

Answer 20

Embryo from mature seed Innoculated with agrobacterium Generate callus CRE excises BBM/WUS construct Select transformed callus with BBM/WUS contruct excised

Answer 21

Single somatic embryos on the surface of Fast-Flowering - induced by Wus2 and Bbmexpression

Answer 22

Crop transformation technologies can lead to higher crop yields, helping to meet the growing demands of a growing population

Answer 23

Genes can be inserted to increase the amount of essential nutrients, such as vitamins and minerals, in crops

Answer 24

Crops can be engineered to express proteins that combat pests, pathogens, and viruses, reducing reliance on pesticides

Answer 25

Increased shelf life banana Wheat with decreased land - single mutant +6% weight, double +11% weight and triple +21% weight Rust resistance BCAT1

Answer 26

Transgenesis refers to inserted DNA originating from a sexually incompatible species Cisgenesis refers to inserted DNA originating from the same or a related species that is sexually compatible

Answer 27

Cisgenes generally include the introns and flanking native promoter and terminator in the same orientation

Answer 28

Most abundant element in the earth’s atmosphere 4th most abundant element in a plant (after C, H and O) Usually the limiting nutrient for plant growth Nitrogen is one of the three major macronutrients found in most fertilizers NPK

Answer 29

N is in amino acids (proteins), nucleic acids (DNA, RNA), chlorophyll, and countless small molecules

Answer 30

Biological fixation through microorganisms (120 Tg N/ yr Industrial fixation through haber bosch process

Answer 31

1909 - Haber-Bosch synthesis of ammonia from N2 1930s - late 1960s - Green Revolution - Breeding programes Population rose from 2 billion in 1900 to 6,148,898,975

Answer 32

Many prokaryotes oxidize NH4+, so soil NH4+ levels are often low Ammonium to nitrate through nitrification

Answer 33

Plants use energy to reduce NO3- for assimilation into organic compounds NH4+ is added to amino acids to create R-NH3

Answer 34

Plant preferences for NO3- vs NH4 + varies by species and depend on the environment - temperature, water, soil pH. But most plants grow best with a mixed supply

Answer 35

Nitrogen fixation is energy demanding Nitrous oxide (N2O) derived from fertilizer is a major greenhouse gas - areas pf nebraska and california with >10 mg/L of nitrate Unhealthy nitrate from agricultural uses pollutes groundwater - creates cyanobacteria/algal blooms which suffoctated aquatic life

Answer 36

Between 60-40% applied N not taken by crop UK farmers apply an average of 190 kg of N based fertiliser per hectare of crop each year 70% of nitrate in inland waters is estimated to come from agriculture N eutrophication costs year-1 in England and Wales estimated £150 million (Defra)

Answer 37

Two main health issues: (i) infant methaemoglobinaemia (blue baby syndrome) (ii) cancers of the digestive tract. But there is evidence of a possible benefit of nitrate in cardiovascular health

Answer 38

Fertiliser Use Efficiency (FUE) % of applied fertiliser recovered by crop

Answer 39

Nitrogen Uptake Efficiency (NUpE) N-uptake/N-available (Nup/Nav)

Answer 40

Nitrogen Utilisation Efficiency (NUtE) Grain yield/N-uptake (Y/Nup)

Answer 41

Nitrogen Use Efficiency (NUE) Uptake efficiency x Utilisation efficiency NUE = Nup/Nav x Y/Nup = Y/Nav

Answer 42

1960 vs 2010 UK = 28% --> 50% China = 86% to 27%

Answer 43

NHI – N Harvest Index (the fraction of N in the grain relative to the total N taken up, usually at harvest)

Answer 44

Matching crop demand and soil supply Supply – N in the soil Demand – the needs of the crop Supply (soil) = Demand (crop) When they done overlap N loss

Answer 45

Grain yield peaks at ~150 Kg/ha 9t/ha However as N fertiliser increases yield doesnt 300 Kg/ha 8.7t/ha Leaching increases overtime 150kg/ha 60kg/ha compared to 300 Kg/ha at 150kg/ha

Answer 46

Early Stem extension requires large about of nitrogen due to large amount of growth Later in life it requires a smaller amount of nitrogen such as ripening (grain development) Late life crop can remobalise nitrogen from elsewhere in plants

Answer 47

Soil available N Rhizosphere – root/soil interface Root architecture Root transporters for uptake

Answer 48

Suction cup lysimeters – soil water sample for lab analysis Soil core sample – lab analysis Plants as indicators Soil sensors

Answer 49

Soil water nitrate concentrations measured under wheat seedlings at 3 depths for 14 hours. Comparing 2 wheat cultivars – Maris Widgeon (low NUpE) and Robigus (high NUpE)

Answer 50

Similar soil [NO3] measured for the two cultivars at mid and base levels Decrease of the [NO3] under the top layer of the ROBIGUS ROBIGUS as bred for better at abosrbing nitrate at higher levels due to more lateral roots at top layer

Answer 51

LoRa Telemetry: Geo-location and live data Sensor is geo-located, data transmitted via gateway to cloud, and live data available on web based system

Answer 52

Traditional fertilizers don’t match nitrogen availability to plant needs. Slow release fertilizers can more closely match plant needs

Answer 53

Coated urea dissolves and releases slowly, but it can be expensive

Answer 54

Bi-cropping with a legume (N2 fixing plant) eg Maize and soybean Co-cropping or growing in rotation with legumes enriches soil N content Cover crops in the autumn in the UK (green manures)

Answer 55

Understanding process as NO3- is most mobile form, negative charge, so understanding how it works and root exudates to alter nitrification keeping more fertiliser as NH4+ less leeching occurs

Answer 56

Inhibitors of bacterial nitrification cause NH4+ to be retained in the soil, leading to less leaching and less N2O production Denitrifying bacteria cultivated in a bioreactor downstream of a fertilized field protect waterways by converting NO3- in runoff to N2

Answer 57

When nitrogen is abundant, plants allocate less biomass to their roots When nitrogen distribution is patchy, roots proliferate in the nutrient rich patches

Answer 58

Plants have specific transporters for NO3-, NH4+, amino acids and urea uptake HATS = high affinity transporters LATS = low affinity transporters

Answer 59

Nitrogen feedback system regulates --> increased uptake in nitrogen transporter can lead to increased efflux maintaining equilibrium

Answer 60

Assimilation of N Remobilization of stored N Measuring crop N status and demand

Answer 61

Crop N status diagnostics: precision agriculture Total-N, P, K, S Unreduced/stored (NO3, PO4, SO4) Metabolites (chlorophyll, glutathione, malate) Gene expression (PCR test)

Answer 62

Testing crops - chlorophyll * Chlorophyll & nitrogen * Growth & yield * Variation: interactions But if measure change in field means too late to get maximum yield

Answer 63

Assimilation - Carbon pools and TCA cycle Remobalisation - Amino acid recycling, photorespiration Incorporation into amino acids and other nitrogen-containing compounds from glutamate and glutamine Glutamine synthetase (GS) is an important ezyme for nitrogen metabolism

Answer 64

Each N atom may cycle through GS many times as amino acids are recycled during growth and senescence and released due to photorespiration

Answer 65

GS1 (GLN1 genes) Cytosolic protein GS2 (GLN2 genes) Nuclear gene, plastid localized protein GS activity is regulated transcriptionally and post-transcriptionally by cell type, light, [NH4+], circadian cycles, plant carbon status etc

Answer 66

GS activity is correlated with nitrogen use efficiency

Answer 67

High remobalisation through GS High levels of uptake through HATs and LATs High assimilation through nitrite and nitrate reductases

Answer 68

Molecular players in N signaling and regulation. Targets for improving NUE (e.g. transcription factors, microRNAs)

Answer 69

The 11th most abundant element in the earth’s crust The 5th most abundant element in a plant The 2nd most commonly limiting nutrient for plant growth

Answer 70

P has roles in cell structure, energy (ATP) and information storage and energy and information transfer

Answer 71

Phosphorus (P) is assimilated and used as phosphate (Pi) which depending on the pH is H2PO4- ,HPO42- or PO43- Acid pH H3PO4 at Alkaline pH PO43-

Answer 72

Plants are part of the global phosphorus cycle Essentially no atmospheric pool of P (very different from N)

Answer 73

Mining and commercial processing accelerates P entry to biosphere Urbanization removes P from terrestrial cycle and accelerates entry to waterways, causing toxic algal blooms (eutrophification)

Answer 74

Phosphate usage has increased dramatically in the past 70 years Some have argued that we are approaching a period of “peak phosphorus” as deposits become depleted, peaking around 1990s 90% of the world’s phosphate rock reserves are found in 5 countries (Morocco 38%, China 27%) With the current rate of phosphate rock exploration and production, the reserves are likely to be depleted in the next 260 years if no new reserves are added

Answer 75

P recovered from human urine alone could replace >20% of P demands (Urine-reclaiming toilet) Human urine is rich in P, and it can be separated from other waste at the point of origin Urine can be applied directly to plants as liquid fertilizer

Answer 76

N & P-rich Wastewater in Cleaner wastewater out Struvite (NH₄MgPO₄·6H₂O) crystals harvested for use as fertilizer

Answer 77

In Sweden this Gotland experiment compared barley fertilized with urine (right) to plants grown without fertilizer (middle) and ones with mineral fertilizer (left) Urine works as a fertiliser

Answer 78

Almost all P is found in the top 50 cm of soil Used to show how much is avaliable? Olsen P (neutral/alkaline soils) – extracted with sodium bicarbonate Bray P (acidic soils) – ammonium fluoride extract

Answer 79

Soil P: Inorganic: Absorbed, precipitated or mineral Organic: Absorbed or humic layers Avaliable P: Soil solution dissovled HPO42- and H2PO41- Soil microorganisms: Convert Soil P to Avaliable P by solubilisation and mineralisation the opposite can also be done via immobalisation

Answer 80

Like nitrate, phosphate can leach through the soil When reaching change point, much higher levels of phosphate in drainage water

Answer 81

Timing of fertilizer application and management of water flow from can decrease the amount of P that enters waterways Chemical and biological processes including algal production can effectively remove P from wastewaters (irrigation or animal feed recycling)

Answer 82

Phosphorus in soil is in the form of immobile, insoluble complexes Cation-phosphate complexes Al-P are relatively insoluble and immobile in soil; these include oxides and hydroxides of Al and Fe Also organic phosphate - plants do not take up organic phosphate Roots growing in 31P-labeled soil. Only P immediately next to roots is taken up

Answer 83

Exudates from free-living and symbiotic microbes also contribute to P solubilization Such as: Low Molecular Weight Organic Acids (LMWOA) - change pH and bind to metal in complex eg malate -> Al-Malate Phytase-producing bacteria (Phytate = C6H18O24P6) Phosphatases (enzymes) convert organic phosphates to avaliable form

Answer 84

Arbuscular mycorrhizal fungi facilitate Puptake in most plants ~80% of plants associate with mycorrhizal fungi; these associations can facilitate P uptake

Answer 85

Reduced gravitropism Increased formation and elongation of lateral roots and root hairs Aerenchyma (air spaces that allow metabolically inexpensive growth)

Answer 86

Many species of the family Proteaceae found throughout the Southern Hemisphere make short-lived “proteoid” or “cluster” roots to facilitate P uptake Cluster roots increase surface area and also root exudation such as phosphateses and proton pump to modify pH

Answer 87

White lupin (Lupinus albus) is a cluster-root producing legume that provides a good genetic model

Answer 88

PHT1 phosphate transporters mediate uptake and transport Most are expressed in roots and other tissues 9 PHT1 genes in Arabidopsis, 13 in rice, 12 in poplar. Some are mycorrhiza inducible

Answer 89

PHT transporters are H+ / PO43- co-transporters that have 12 membrane spanning domains

Answer 90

Proteaceae show metabolic adaptions to Pimpoverished soils such as very efficient use Ribosomes (rRNA) are the major form of organic P. Proteaceae maintain a very low copy number of ribosomes, yet are photosynthetically efficient Proteaceae also show delayed greening; ribosomes first promote growth, then chloroplast maturation

Answer 91

Increase in Pi recycling and remobalisation Reduced rate of photosynthesis Increased sugar concentration Increased anthyocyanin accumulation Reduced shoot growth

Answer 92

Increased Pi uptake, transport and translocation Increased secretion of organic acids and acid phosphatases Changes in metabolic pathways Establish plant-mycorrhizal fungi symbiosis

Answer 93

PHO1 is a transporter that moves Pi into xylem for transport to the shoot PHT transporters take P PHO2 is an E2 ligasethat targetstransporters for proteolysis

Answer 94

In pho1 mutants, too much Pi accumulates in the root and too little in the shoot In pho2 mutants, too much Pi accumulates in the shoot and too little in the root; transport is out-of-control

Answer 95

P uptake & transport are regulated by local and systemic signals: Strigolactones - Establishment of plant – mycorrhizal fungi symbiosis PHR1 (transcription factor) - PHT1 transporters, Phosphatases, organic acid synthases, IPS1 inhibits miR399 which inhibits PHO2 and PHO1

Answer 96

Can bind to important metal like iron Phosphate can be stored as ‘phytate’. In seeds phytate can make metals like iron and zinc less ‘available’ for uptake in the gut.

Answer 97

Yes Same advantages (Easy to measure, Sensitive to supply, Responds quickly) and problems (once problem detected too late)

Answer 98

6 nM in wheat leaves

Answer 99

Rhizosphere (exudates, acidification, enzymes) Uptake systems (transporters, regulation) Root architecture (root hairs, laterals, surface, depth gravitropism, penetration) Assimilation (capture, stores, translocation, symbioses) Symbioses (fungi - mycorrhization for P; phosphate solubilising bacteria)

Answer 100

Modifying regulators of P signaling network Releasing Pi from insoluble pools (through organic acid extrusion, proton pumping, and phosphatases) Optimizing root architecture Enhancing high affinity uptake (PHT1 transporter) Success has been mixed, internal regulation increased expulsion

Answer 101

Selection for root architecture traits can lead to increased P uptake Select for cultivars on low P soil with top heavy root system

Answer 102

The Pup1 (Phosphate Uptake 1) major QTL was identified in ausvariety rice adapted to poor soils Eventually this was revealed to encode a protein kinase PSTOL1 not present in other rice genomes Overexpression of PSTOL1 leads to enhanced root growth

Answer 103

The effect of strigolactones (SLs) on the host plant, the arbuscular mycorrhizal (AM) fungus and its endobacteria during the establishment of AM

Answer 104

A1- enhancing Pi transport from source to sink during the vegetative growth (PHT1, PHT2 families) A2 - preferential allocation of Pi to photosynthetic leaf cells A3 - Reducing P transport from source to sink during reproduction stage (SPDT, PH01:2)

Answer 105

B1 - Phosphate recycling from vacuole B2 - Pi liberation from organic P pools

Answer 106

Membrane remodelling and liberating Pi from Lipid-P (LPA, LPC, LPD) Phosphotases to liberate Pi from Pi-esters (PAPases, PPsPase) Economically liberating and using Pi from Pi-esters (RNases, DNases Quantifying the contribution of P fractions allocation to PUE

Answer 107

Phosphite - [HPO3]2− A few bacterial strains that are capable of oxidizing phosphite to produce phosphate but plants cannot.

Answer 108

Transgenic Arabidopsis plants that express a phosphite oxidoreductase can use phosphite as a P source A.t with ptxDAt-3,5,7 grow they can but WT can't grow showing additonal herbcide function

Answer 109

In phosphite environments the tobacco grew well but the weeds did not Weeds and tobacco grown together in normal soil saw weeds outgrow Both seperately both grew well

Answer 110

Soil mobility differs – NO3- mobile; phosphate and NH4 + immobile Symbioses – bacteria and fungi Root architecture and transport important Need to match soil supply to crop demand Measuring supply/demand – for ‘precision agriculture’