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3s2: Plant biotechnology > Week 7 > Flashcards

Week 7 Flashcards

1
Q

Why is starch important?

A

Starch is the major calorific component of many of our staple crops.
Top 16 cops by harvested areas. 9 are starch, including top 3, wheat, maize and rice
Other important sources of starch include almost all other cereal crops (rye, oat), root and tuber crops (yams), and bananas/plantains.

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2
Q

What is the history of starch crops?

A

Ancient Egyptian grew emmer wheat
Stone tool from hundreds of thousand years ago still have starch granules

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3
Q

How is important is starch?

A

European starch industry processes about 20-25 million tonnes of raw materials per year for industrial uses of starch such as paper, solution thickening and beer

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4
Q

What is the evolutionary groups of carbohydrate biopolymers?

A

Plants and algae store starch as amylose (~1,000 glucose units) and amylopectin (~100,000-1,000,000 glucose units)
Bacteria, fungi and metazoans store glycogen (~50,000-100,000 glucose units

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5
Q

What is the structure of starch?

A

Starch is composed of two glucose polymers:
Amylopectin (highly branched) and amylose (mostly linear).
α-1,4-glucosidic bonds in linear chains, α-1,6-glucosidic bonds at branches

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6
Q

What is an overview of amylopectin?

A

Amylopectin has a similar chemical structure to glycogen, but the latter has shorter chains and more branches.
Starch is insoluble while glycogen is soluble

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7
Q

What is an overview of amylopectin and amylose assembely into insoluble starch granules?

A

The structure of amylopectin permits the formation of double helices between adjacent chains.
Helices pack together to give crystalline lamellae, which exclude water

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8
Q

What is an overview of starch insolubility?

A

Formation of an insoluble, semi-crystalline starch granule.
Starch is an ideal storage carbohydrate because it is compact, inert, and osmotically inactive

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9
Q

What is an overview of starch consumption?

A

Most plants store starch in leaves.
The conversion of starch to sucrose during the night allows plants to grow in the dark.
Starch in storage or reproductive organs is stored over longer periods of time. Starch degradation provides energy to fuel the growth of sprouts/seedlings

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10
Q

What is an overview of Starch provides both short-term and long-term energy storage?

A

Over night - leaves
Over months - tubers (like potatoes)
Over years - seeds

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11
Q

What happens when a plant cannot synthesise starch?

A

Wildtype vs pgm mutant
24hr day - wt and pgm level, with mutant overtaking
12h day vs 12h night - WT starts similar but overtakes pgm quickly over time
7hr day vs 17hr day -W

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12
Q

Why is monitering starch synthesis important for experiments?

A

Mutants that cannot synthesise starch in leaves starve during the night.
Biotechnological modification of starch needs to consider the role of starch in plant metabolism.
Approaches need to avoid detrimental effect on growth

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13
Q

What is the mechanism for leaf starch synthesis from the products of photosynthesis?

A

Fructose-6-P <-(Phosphoglucoisomerase (PGI) -> Glucose-6-P <- Phosphoglucomutase (PGM) -> Glucose-1-P - ADPGlc pyrophosphorylase (AGPase) -> ADP-Glucose –> Starch

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14
Q

What are the origins of things in leaf starch synthesis?

A

CO2 fixed in the Calvin-Benson cycle in leaves produces Fructose-6- phosphate.
Fructose-6-P is used to produce ADPglucose, the substrate of starch synthesis.
AGPase catalyses the first committed step of starch biosynthesis.

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15
Q

Outside of chlororplast where else can starch be made?

A

In non-green/heterotrophic tissues, starch is made in nonphotosynthetic plastids specialised for starch storage (the amyloplast) using imported sugars (sucrose)

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16
Q

Why is AGPase important?

A

AGPase catalyses the first committed step of starch biosynthesis
The hydrolysis of the pyrophosphate renders the AGPase reaction irreversible
ADP-Glucose is used by starch synthases to elongate polymer chains

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17
Q

Why is ADP-glucose a good glucocyl donor?

A

Its an activated glucose
Adenosine molecule linked to the reactive carbon of the glucose which happens through a double phosphate bond, which when broken to join starch polymer, gives off a large amount of energy

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18
Q

Why is AGPase reaction irreversible?

A

Glucose-1-P to ADP-Glucose requires ATP releasing Pyrophosphate (P-Pi)
Pyrophosphatase cleaves P-Pi to 2xPi releasing a large amount of energy
Cant be rejoined therefore irreversible

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19
Q

Why is AGPase located?

A

In leaves, AGPase is located in the plastids.
In cereal endosperm, AGPase is located in the cytosol.
In this case, ADP-glucose must be transported into the plastid

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20
Q

What is an overview of amylopectin synthesis?

A

Amylopectin synthesis requires the orchestrated action of at least three different classes of enzymes

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21
Q

What are the steps for amylopectin synthesis?

A

Starch synthases (SS) - elongate polymers by ADP-glucose
Branching enzymes (SBE) add new branches
Debranching enzymes (DBE) or Isoamylases (ISA) remove misplaced branches

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22
Q

What happens if starch synthases are mutated?

A

All plants have at least four isoforms of starch synthase (SS1, SS2, SS3 and SS4), which specialise in different parts of amylopectin formation.
Arabidopsis mutants lacking ss isoforms still make starch, but the starch has highly aberrant amylopectin structure

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23
Q

What is an overview of chain length distrubution?

A

“Chain length distribution” = amylopectin structure
Wild type ss1 mutant ss2 mutant ss3 mutant
ß Mutants have altered amylopectin structure (lower degree of polymerisation)

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24
Q

What is mendels relationship with starch in peas?

A

Mendel’s wrinkled pea mutant is defective in starch branching enzyme
The R locus encodes one of two starch branching enzyme
isoforms in pea.
Starch in wrinkled pea (rr) has defective SBE activity,
which results in a large decrease in total starch content

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25
Q

What is the difference between wrinkles (sweet) pea and round pea?

A

Wrinkled (sweet) pea – most human consumption
Very low starch content
High sugars (sweet)
Starch that does accumulate is resistant to digestion

Round pea – mostly used as feed
High starch content
Low sugars (not sweet)

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26
Q

What is similar between amylopectin and glycogen biosynthesis?

A

Starch synthases (SS) = Glycogen synthases
Branching enzymes (SBE) = Glycogen branching enzymes
Debranching enzymes (DBE) or Isoamylases (ISA) = No comparable step

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27
Q

What is an overview of mutants that produce glycogen instead of starch?

A

Arabidopsis mutants deficient in the debranching enzyme involved in starch synthesis produces ‘phytoglycogen’ rather than starch
In chloroplasts thereare soluble phytoglycogen not noticable starch granules

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28
Q

What is special about Cecropia peralta?

A

Cecropia forms a symbiotic relationship with ants.
Expression of debranching enzyme is turned off in feeding bodies to provide the ants with glycogen

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29
Q

Where does amylose synthesis take place?

A

Amylose synthesis takes place within the granule
Site of amylose synthesis Fills space inside the granule in amorphous regions
Site of amylopectin synthesis - Growth at the surface of the granule

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30
Q

What is an overview of GBSS?

A

Granule Bound Starch Synthase - a specialized isoform of starch synthase for amylose biosynthesis

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31
Q

What is an overview of Amylose-free starch generation?

A

‘Waxy’ maize mutants produce amylose-free starch. The mutated gene is GBSS.
Amylose-free starch leads to thicker, stickier textures

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32
Q

What is an overview of Amylose-free starch crop production?

A

Cultivated for centuries - positive selection for the trait in Asia.
Waxy maize grown on a commercial scale (mostly in the US). Desirable starch quality for many industrial uses of starch with almost no yield penalty.
Waxy barley and rice also originate from Asia, and are mostly grown there.
In rice, the waxy trait is known as “glutinous”

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33
Q

What is an overview of well known starch biosynthesis mutants?

A

Indica vs. Japonica rice - Japonica rice is defective in a
gene encoding SS2 – its altered amylopectin structure makes it stickier when cooked.
Wrinkled (sweet) pea is defective in a gene encoding SBE – its low starch makes it sweet
Sweet corn is defective in a gene encoding ISA – its low starch makes it sweet, and it contains phytoglycogen
Glutinous rice is defective in a gene encoding GBSS. The amylose–free starch makes it extremely sticky

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34
Q

How do we improve the quality of starch crops for certain industies?

A

Thickeners and emulsifiers - optimise gel consistency and avoid retrogradation amylose-free starch
Finishes and adhesives - requires clear, sticky gels and amylose-free starch
Biodegradable plastics and coating - optimise durability and high-amylose starch

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35
Q

How do we improve the quality of starch crops for foods?

A

Baking - optimise starch digestibility for health benefits and high-amylose starch
Brewing - optimise starch digestion during malting and consistency in granule size allows uniform digestion
Pasta - optimise water absorption during cooking by low amylose content and smaller granules

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36
Q

What is an overview of the uses of chemical modifications by the starch industry?

A

Despite being able to modify some physico-chemical characteristics of starch in planta, many applications still require extensive chemical modification to achieve desirable starch characteristics.
More approaches to engineer starch using genetics may reduce our reliance on expensive and environmentally unfriendly modifications

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37
Q

What is an overview of gelatinisation?

A

Gelatinisation occurs when starch granules are heated in the presence of water.
Granules swell and eventually burst, forming a viscous gel. Crystalline structure is lost during this process.
Eventually, the polymers recrystallize over time. This is called ‘retrogradation’.

38
Q

What is an overview of amylose-free potatoes genetic editing?

A

BASF developed ‘Amflora’ – targeted GBSS using antisense-mediated silencing.
Avebe markets a non-transgenic amylose-free potato containing mutations in GBSS.

39
Q

What is an overview of acceptance of BASF developed amflora?

A

Marketed towards industrial users of starch (mainly the paper industry), but faced lack of consumer acceptance.
First GM crop to be approved by the EU (2010), but later withdrawn.

40
Q

Why is altering starch properties important for potentially improving human health?

A

Obesity costs society £27 billion
Obesity costs NHS £6.1 billion
More money on obesity related diseases than police, fire and judicial system combinedt

41
Q

Why could altering starch properties important improve human health?

A

Resistant starch escapes digestion in the stomach and small intestine:
* Starch encased in cell walls evades digestive enzymes.
* Starch with high amylose content is resistant to digestion

Resistant starch - Bacterial fermentation, Short-chain fatty acids, Improved gut health and insulin production
Normal starch - Rapid digestion – rise in blood glucose

42
Q

What is the difference between control and high amylose potatoes?

A

Control and high amylose, starch high concnetrated in small area before boiling
When boiling starch every where in control but not in high amylose as granules do not completely gelatinise

43
Q

Why does high amylose potatoes resist digestion?

A

High-amylose starches are resistant to digestion, due to the tight crystalline packing of amylose

44
Q

What are the mechanisms for higher amylose content?

A

General suppression of amylopectin synthesis
Formation of longer ‘amylose-like’ chains on amylopectin due to lessfrequent branching

45
Q

What is an overview of starch granule morphology?

A

Vast diversity in starch granule morphology among different cereal grains
BIMODAL (e.g: wheat, barley)
COMPOUND (e.g: rice, most grasses)
UNIMODAL (e.g: maize)

46
Q

What is an overview of bimodal starch granule morphology?

A

In amyloplast large starch granule then 15 to 20 days later secondary B granules form in protusions

47
Q

What is an overview of compound starch granule morphology?

A

In amyloplast all form together at same time, then fuse together and fall apart creating polygonal structure

48
Q

What is an overview of unimodel starch granule morphology?

A

Just one large starch granule per amyloplast

49
Q

What is an overview of genome editing of starch granule formation?

A

Discovered factors influencing different starch granule morphologies in cereal grains. Can now engineer all morphology types in wheat
We are now testing the effect of these modifications on different applications (e.g., bread, pasta)

50
Q

What are the genes responsible for different starch morphologies?

A

BIMODAL - (WT)
COMPOUND - (ss4, bgc1)
UNIMODAL - (bgc1, phs1)

51
Q

What is a compatible interaction?

A

Compatible interaction
Plant is ‘susceptible’, pathogen ‘virulent’

52
Q

What is an incompatible interaction?

A

Incompatible interaction
Plant is ‘resistant’, pathogen ‘avirulent’

53
Q

What is the interaction matrix?

A

The interaction matrix is a tool for understanding potential reactions between pathogens raves and plant cultivars showing race-specific interaction

54
Q

What was the overview of the history of race-specifc resistance?

A

Using classical Mendelian genetics of both plant (flax) and pathogen (Melampsora lini - flax rust) H. H. Flor, working in the 1940s
Created the gene-for-gene hypothesis

55
Q

What was the outcome of the gene for gene hypothesis?

A

1) Resistance is dominant to susceptibility in the plant

2) Avirulence is dominant to virulence in the pathogen

3) For each gene for resistance (R gene) in the plant there is a corresponding gene for avirulence (Avr gene) in the pathogen

4) Interactions are only incompatible when the genome the host contains a resistance gene and the pathogen contains the corresponding avirulence gene

56
Q

What is an overview of receptor-ligand model for ETI?

A

Avrs are effectors plants recognise
Where a specific R protein binds to a specific AVR gene triggering resistance
No matching pair no resistance

57
Q

What is an overview of guard model for ETI?

A

AVR gene binds to effector target
R protein detects changes to effector target triggering resistance
No matching pair no resistance

58
Q

What are the different types of disease resistance?

A

PTI: PAMP-triggered immunity
ETS: Effector triggered susceptibility
ETI: effector triggered immunity

59
Q

What is the order for disease resistance in plants?

A

Through evolutionary time
PTI inital
Effectors overcome PTI triggering ETS
ETS forces an evolutionary response of ETI
ETI is then overcome, forcing evolution to respond

60
Q

What are PAMPs and examples?

A

PAMPs: pathogen associated molecular patterns: Conserved pathogen molecules E.g: bacterial flagellin, bacterial cold-shock proteins, bacterial elongation factor Tu (EF-TU) and fungal chitin

61
Q

What occurs when PAMPs are detected?

A

PTI is initiated when PAMPs are recognized by transmembrane receptors called pattern recognition receptors (PRRs), e.g : FLS2 (binds flagellin), EFR (binds EF-TU) and CER1 (binds chitin)

62
Q

What is an overview of FLS2?

A

FLS2 (flagellin sensing 2) is a transmembrane receptor kinase that binds to a 22 amino acid peptide from flagellin. The 1173 amino acid protein has an extracellular domain with 28 leucine rich repeats (LRRs) and an intracellular protein kinase domain

63
Q

What is an overview of fls2 mutants?

A

fls2 mutants exhibit enhanced susceptibility to bacterial pathogens such as Pseudomonas syringae pv tomato (Pst) up to 10x greater depending on leaf age

64
Q

What is structure of FLS2?

A

N-terminus: signal peptide, LRRs, Transmembrane domain and Cytoplasmic protein kinase domain

65
Q

How does FLS2 detect flagellin?

A

In the absence of flg22, FLS2 and BAK1 interact with BIK1, and BAK1 interacts with and phosphorylates BIR2.

Flg22 binds to FLS2, the BAK1-BIR2 complex dissociates and FLS2, flg22 and BAK1 form a heterodimeric complex. Subsequently, BAK1 phosphorylates BIK1 and BIK1 phosphorylates both FLS2 and BAK1.

66
Q

What the relationship between stomata and infections?

A

In response to bacteria plants close their stomata
In response to virulent bacteria, but these bacteria have evolved virulence factors that induce reopening.

67
Q

What is the T3SS?

A

The type III secretion system (T3SS) delivers effector proteins into host cells through a needle-like apparatus that spans bacterial envelope, whereby mediating the bacterial invasion of host cells

68
Q

What makes the T3SS?

A

Several Hrp proteins form a pilus through which effectors (including Avrs) are translocated

69
Q

What are examples of T3 effectors and their function?

A

AvrPto inhibits the function of FLS2
Hopl1 inhibits SA

70
Q

What is an overview of the TALE family?

A

AvrBs3, PthA, AvrXa7 and related proteins from Xanthomonas species make up the TALE family
TALE: transcription activator-like effectors
Exhibit sequence-specific DNA binding
Act as transcriptional activators in the plant cell nucleus

71
Q

What is an overview of TALE binding?

A

Each 33-35 amino acid repeat binds a single nucleotide. Specificity is determined by a two amino acid sequence. HD binds C, NN binds G, NG binds T, NI binds A

72
Q

What are exmaples of TALE effectors?

A

Xcv AvrBs3 targerts BR3 gene in pepper a resistance gene
Xoc Tal-C1c targets OsHEN1 a sRNA biogenesis gene
Xoo PthXo1 targets Os8Ns a nodulin MtN3 family

73
Q

What is a key function of AvrPtoB in HR response?

A

AvrPtoB suppresses HR and cell death in yeast
Suggests that the suppression of HR is the virulence function of AvrPtoB

74
Q

What is a key function of AvrPtoB in degredation?

A

AvrPtoB causes degradation of FLS2
AvrPtoB is an Ubiquitin E3 ligase
Suggests that suppression of FLS2 is the virulence function of AvrPtoB

75
Q

What is an overview of Magnaporthe oryzae effector Slp1?

A

Rice recognises chitin, a fungal PAMP: chitin oligosaccharides are bound by chitin elicitor binding protein (CEBiP), initiating defence (=PTI)
Rice blast fungus (Magnaporthe oryzae) Secreted LysM Protein1 (Slp1) binds to chitin and suppresses chitin-induced plant immunity

76
Q

What is an overview of NLR (Nucleotide binding LRR) N protein?

A

Confers resistance to Tobacco Mosaic Virus (TMV)
1144 amino acids
No potential signal peptide or transmembrane domain
Member of a multi-gene family

77
Q

What is an overview of NLR protein N structure?

A

Nucleotide binding site (NBS) in exon 2 encoded domain
C-terminus contains 14 leucine rich repeats
TIR domain at N terminus: Toll and human interleukin 1 receptor (HIL1R) homology

78
Q

What are different classes of NLR R proteins?

A

TIR-NLRs (TNLs) such as M (nemotode) and RPP5 (downy mildew)
CC (coiled coil)-NLRs (CNLs) such as RPS2 (Pseudomonas syringae pv. tomato) and I2C1 (Fusarium oxysporum, vascular wilt disease)

79
Q

What is an overview of classes of non-NLR R proteins?

A

LRR + TM = Cf2/4/5/9 (tomato)
KRR + TM + S/T Kinase = Xa21 (Rice)
S/T kinase = Pto
TM + CC = RPW8

80
Q

What is RIN4?

A

RPM1 interacting protein (identified by yeast 2-hybrid). Interacts in planta with RPM1 and AvrRPM1 and AvrB

81
Q

What is the function of RIN4?

A

RIN4 is required for RPM1-mediated HR and RPM1-mediated Resistance
The AvrRPM1/B-RIN4-RPM1 system is an example of an R protein guarding an effector target

82
Q

What is an overview of NLR interaction with effector target: ZAR1-RKS1- PBL2UMP interaction?

A

PBL2UMP modified by AvrAC interacts with the preformed ZAR1-RKS1 complex triggering ADP, ATP exchange, pentamerisation and formation of the active resistosome

83
Q

What is an overview of ZA1 gene immunity?

A

ZAR 1 is a CC NLR that confers resistance to Xanthomonas campestris pv. campestris

84
Q

What is an overview of ROQ1-XopQ?

A

ROQ1 is a TIR NLR that confers resistance to Xanthomonas euvesicatoria expressing XopQ
ROQ1 interacts directly with Type III effector XopQ

85
Q

What is an overview of ROS produciton for immunity?

A

Signalling includes synthesis of reactive oxygen species (ROS) that cause programmed cell death

86
Q

What is an overview of HR response?

A

Programmed death of plants cells (=hypersensitive response, HR). HR deprives obligate pathogens of the living cells they require

87
Q

What are proteins highly produced in ETI?

A

Pathogenesis-related (PR) protein gene expression: PR proteins are antimicrobial, incl. glucanases and chitinases
Phytoalexin biosynthesis gene expression - Phytoalexins are low molecular weight antimicrobials

88
Q

What is the immune network theory?

A

Pathogen molecule is detected by receptor which can have multiple coreceptors triggering downstream components
This contrasts with the simplified binary view of the gene-for-gene hypothesis.

89
Q

What are examples of immune network theory?

A

NRC2 promotes signalling in response to Bs2 (bacterial AvrBS2) and Gpa2 (Nemotode)
Bs2 also promotes signalling in the NRC3 and NCR4 coreceptors

90
Q
A

3s2: Plant biotechnology (9 decks)

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