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Flashcards in lecture 29 Deck (20)

What is evidence that auxin is a morphogenetic trigger?

- mutants lacking basal structures are defective in auxin signalling (mp/bdl mutants)
- auxin accumulates in the basal region of the embryo prior to hypophysis formation


What is the difference between a morphogen and a morphogenetic trigger?

- morphogen
- cell fate determined by distribution of a morphogen
- diffusion gradient
- different cell fates at different levels
- the morphogen is instructive along the entire field of cells

- morphogenetic trigger
- instructive in only a small group of cells
- e.g. only when in high concentrations
- these cells acquire a different fate
- but the trigger is not instructive in cells with lower concentrations


What is the concept of master regulators?

- cell division: no distinction between cells in the field
- patterning: specific sets of transcription factors are activated according to positon
e.g. TF1 in position 1 and 2, TF2 in 3 and 4, TF3 in 5 and 6
- differentiation: cell/tissue specific programme of gene expression: e.g. TF1 --> head genes, TF2 --> thorax genes, TF3 --> abdomen genes

- master regulators orchestrate specific patterns of gene expression
- this is true of animals, is it true of plants?


What are master regulators of axial cell fate?

- Plethora (PLT) genes encode transcription factors that function to promote root formation
- phenocopy of mp mutants - lack of hypohysis
- need to inactivate all members of the family because of redudancy
- to test whether the PLT genes are master regulators of cell fate, generate transgeneic plants in which Plt genes are mis-expressed
- homeosis - conversion of shoot to root
- this evidence suggests that plethora genes are master regulators

- would expect the plethora genes to activated by auxin, and by mp


Does PLT act downstream of auxin/Mp?

two lines of evidence suggest PLTs are regulated by auxin
1. loss of PLT expression is observed in mp mutants
2. PTL gene activity is responsive to exogenously applied auxin (IAA/NAA)
- increase in plethora expression after 24 hours

- PLT expression accumulates in the hypophysis precursor cell in wt embryos at globular stage
- slightly more broadly in the adjacent basal embryonic cells

no expression in mp mutants therefore plethora expression is absolutely dependent on monoptrous activity therefore downstream


What is the current model for embryonic root patterning?

- auxin --> monoptrous --> plethora
- BUT Mp is not actually expressed in hypohpysis precursor cell
- basal embryo cell just above hypophysis precursor
- auxin --> bdl inhibition --> mp upregulates PIN --> PIN genes encode auxin efflux transporters --> movement of auxin in hypophysis precursor cell

- if this model is correct, then mp mutant prevents auxin from accumulating in precursor cell since it doesn't trigger expression of PIN proteins
- could we then rescue cell adding auxin to hypothesis ? no.
- this model has something missing

- exogenously applied auxin does not rescue mp mutants
- must be a MP-dependent signal that moves from the basal cell into the hypophysis precursor

- this has possibly been identified


What is organogenesis?

- leaves (vegetative organs) arise in the periphery of the shoot apical meristem
- arranged in a regular and predictable pattern - phyllotaxy
- most common arrangement is spiral (~137.5º between adjacent organs)
- new leaves tend to arise in the first available space
-- i.e. in a region furthest from existing leaves
-- greatest space
- suggests that growing leaves are exerting some kind of inhibition on newly arising leaves --> lateral inhibition

- if you surgically remove a leaf, the position of the new leaf shifts, indicating that the removed leaf was exerting some kind of inhibtion


How do we know that auxin promotes organ formation?

- mutants with defects in auxin production, transport or perception display changes in phyllotaxy e.g. pin-formed1 (pin1) mutants
- tells us that auxin promotes organ formation
- exogenous application of auxin restores organ formation from the pin1 mutant apex


What is field theory of phyllotaxy?

- organ formation requires auxin accumulation
- developing organs deplte auxin from surrounding regions
- new organs can only form in regions of least depletion


What is PIN localisation in the SAM?

- PIN1 accumulates in membranes facing future site of organ initiation
-- auxin accumulates at future sites of organ formation and drained from surrounding tissue
-- net sink
- PIN1 polarity reverses
-- auxin flows to new sites of organ formation
-- net source

- sequential auxin peaks in the SAM trigger organ formation
while there is a gradient of auxin, it is only very high levels that trigger organ formation


Give a summary of plant morphogens

- positional information used extensively in plant development
- no evidence for maternally encoded morphogens
- auxin has morphogen-like qualities
-- gradient in tissye
-- acts directly on cells via activation of ARFs
-- determines cell fate according to concentration (female gametophyte)
- auxin frequently behaves as a morphogenetic trigger
-- hypophysis formation
-- phyllotaxy


What is control of post-embryonic cell fate re: the flower?

- floral meristems arise from the inforescence meristem
- produce concentric whorls of organs with distinct identities
- determinate: produces limited number of organs
- genetic analysis identified homeotic mutants - where one floral organ is replaced with another
- three classes of mutant (A, B, C) - affects organs in TWO adjacent whorls

wt flower
whorl 1 - 4 sepals
whorl 2 - 4 petals
whorl 3 - 6 stamens
whorl 4 - 2 carpels


What is a floral meristem?

modified axillary bud
as a meristem able to produce organs along its periphery

produces whorls instead of spirals
ceases activity once 4th whorl is produced


What is a class A mutant flower?

Wh 1: sepals --> carpels
Wh 2: petals --> stamens
Wh 3: stamens
Wh 4: carpels

apetela2 (ap2) flower
apetala1 (ap1) flower


What is a class B mutant flower?

Wh1: sepals
Wh 2: petals --> sepals
Wh 3: stamens --> carpels
wh 4: carpels

apetala3 (ap3) flower
pistillata (pi) flower


What is a class C mutant flower?

wh 1: sepals
wh 2: petals
wh 3: stamens --> petals
wh 4: carpels --> sepals


adamous (ag) flower


How does the ABC model predict mutant phenotypes?

- we know that the mutations in these genes are affecting two adjacent whorls
- class A: 1 and 2, therefore activity spans 1 and 2/class A active in whorls 1 and 2
- class B: 2 and 3, petal and stamen
- class C: 3 and 4

overlaying this information you can see:
- sepals are regulated only by class A activity
- petals Class A and B (in combination)
- stamens: B and C (in combination)
- carpels: C only

combinatorial model

but there is another component to this model
- if you have a mutation in a class A gene we know that you affect organ identity in whorls 1 and 2
- what happens is that whorl 1 should be forming sepals but instead forms carpels, whorl 2 should be forming petals but it forms stamens
- this suggests that C class genes are now affecting whorls 1 and 2
- therefore Class A genes must antagonise class c genes from being functional in whorsl 1 and 2 normally
- same is true in class C mutants
- mutual repression of these two classes of genes

A mutant:
- Ca, St, St, Ca

B mutant:
- Se, Se, Ca, Ca

C mutant:
- Se, Pe, Pe, Se


What is evidence supporting the ABC model?

- look at the expression of ABC genes: in situ hybridisation

- Apetala 1 expressed in sepals and petals
- Apetala 3/Pistillata expressed in petals and stamens
- Agamous in stamen and carpel

- se-pe-st-ca-ca-st-pe-se

- three concentric overlapping regions of gene expression established

model predicts phenotype of double mutant combinations
- class b and c mutants: a activity spreads to 3 and 4, alone, sepals in all four whorls
- highly predictive

model predicts phenotypes arising when homeotic genes are expressed ectopically in the flower:
- transgenic experiments
- make B expressed in 1 and 4
- pe-pe-st-st


What is the combinatorial action of ABC genes?

- predict that ABC genes encode master regulators
- MADS transcription factors
- bind to promoters of target genes as tetramers
- bind to genes that express sepal, petal, stamen and carpel identity respectively
- identity of some of the proteins in the complex have not yet been established

Ap1x2 + unknownx2 = sepal promoter

ap1+ap3+PI+MADS box protein = petal

AG + PI + AP3 + MADS = stamen

AGx2 + MADSx2 = carpel


Give a summary of plant development

- rigid cell wall and no cell migration
-- form generated by regulating the plane of cell division and direction of expansion
- asymmetric distribution of auxin establishes pattern
-- acts as morphogen or morphogenic trigger
- most development occurs post-embryonically
-- meristems construct reiterative body plan
- cellf ate determined by spatially restricted master regulators
- identity of positional signals and regulatory genes is different from animals - MADS-box genes vs Hox genes