plant development and environmental regulation of development

Question 1

plants, like Arabidopsis, show polarity super early - how?

Answer 1

Earliest stages of embryogenesis in arabidopsis

When heart stage is reached, there’s already definitive apical-basal polarity and bilateral symmetry

Can also see the shoot and root meristems from which all plant material is derived. this all requires asymmetric divisions

Question 2

what are some restrictions of plant cells, that animal cells don’t have, when it comes to development?

Answer 2

no cell movement, rigid cell walls, shape is determined by cell division plane and cell expansion

Question 3

in plant embryogenesis, why is the embryo proper called ‘1-cell’, but looks like 2?

Answer 3

only the upper cell goes on to form the embryo proper. The basal cell forms a structure called the suspensor, which connects the embryo to maternal tissue in the ovule

Question 4

using Arabidopsis as our model for dichotomous plants (meaning two cots at the heart stage), what are the organs of a flower?

Answer 4

Four organs, arranged in concentric ‘whorls’

All organs derived from an inflorescence meristem

Outer to inner - sepals, petals, stamen, carpals

Question 5

what are three mutants affecting flower organs and their phenotypes?

what’s the first question then asked about these mutants?

Answer 5

Apetala1 → only carpels and stamen
Pistillata → only sepals and carpels
Agamous → only sepals and petals

SO - are these deletions of organs, or replacements?
ANSWER - these are replacements. In each mutation, two whorls have had the correct organ replaced, while the other two remain ‘correct’

Question 6

explain how AP1, PI and AG can explain organ expression of a flower

Answer 6

the expression domains of the three genes create four unique environments, each causing the production of a flower (light be good to look at a picture)
In situ hybridisation confirmed that these are the expression domains, e.g. apetala1 is expressed in whorl 1 and 2 (stuck AP1 promoter to GFP)

Sepals could be specified by a domain in which only AP1 is expressed

Petals require both AP1 and PI function

Stamen require PI and AG

Carpels require only AG expression

note - when thinking about this, remember it’s mirrored

Question 7

what is the ABC model?

important detail?

Answer 7

basically already explained, three domains, i.e. a different gene expressed in each one, some organs occur when just one of these genes is present, some require two of them/an overlap…

A domain (the outer whorls) is where you find sepals and petals

B domain - moving in, is where you find petals and stamen

C domain - central, is where you find stamen and carpels

so:
Sepals (A)
Petals (A+B)
Stamen (B+C) Carpels(C)

A and C must antagonise each other and restrict their respective expression domains

Question 8

what genes are expressed in the A, B and C domains, and what happens if you have e.g. an AP1 mutant/ A domain gene mutant?

Answer 8

A domain = apetala1 and 2
B domain = apetala 3 and pistillata
C domain = agamous

A mutant / no A gene - no A to antagonise C, so C expressed everywhere
Just A (whorl 1) replaced by just C (whorl 4), so sepals become carpels
A+B → C+B so petals become stamen
Whorl 3 Stamen (B+C) and whorl 4 carpals (C)aren’t affected

Question 9

what are the A, B and C genes?

Answer 9

Homeotic genes

MADS transcription factors (not homeodomain TFs)

Have a 50-60 Aa DNA binding domain

Question 10

what is the ABCE model?

Answer 10

Sometimes referred to as this
E represents the SEPALLATA genes

These genes are only expressed in developing floral meristem/floral specific, so overexpression of A or B or C genes in e.g. the leaves or roots, wont turn the leaves or the roots into e.g. petals, because sepallata genes are required to form complexes with ABC genes before they can dictate organ identity

Question 11

no B gene?

Answer 11

the A+B domain is now just A, so petals become sepals

the B+C domains now just C, so stamen become carpals

normal carpels are still there (just C), as are normal sepals (just A)

Question 12

no C gene?

Answer 12

C isn’t present to antagonise A, so A is produced all over

C domain now just A, so carpels replaced by sepals

B+C now B+A, so stamen become petals

Question 13

why is environmental regulation of development studied in plants more than animals?

Answer 13

while animal development is regulated by the environment, e.g. temperature can regulate sex determination (common in amphibians), or human height and nutrition

Plants -
They can’t move, so have to adapt (and their environment is easy to set as well)
Changes that occur are more dramatic (however - often you get repetition of pattern - more branches but the same pattern?)
Much more ‘amenable to genetic analysis - response to environment is easier to quantify, and mutational analysis is easier to perform

Question 14

light as a language - three ways?

Answer 14

Photoperiod - day length

Quantity - the amount of light reaching a surface

Quality - the balance of different wavelengths

Question 15

bit of background on light - why is the sky blue, clouds white, sunrise/sunset?

Answer 15

Blue and violet light is shorter, so more scattered by Earth’s atmosphere (O2 and N2), which is why we see the sky as blue

Clouds - made of bigger particles like water - larger than WLs of light and scatter it all equally, hence the white)

sunrise/set - sun angle drops - light travels further/through more atmosphere before reaching us, even more blue is scattered, hence the red/orange colour of the sky we get. Also get an increase in far-red over red light but this is due to refraction

Takeaway - we see changes in light quality across the day or seasons (winter sun is at a lower angel in the sky)

Question 16

how can light wavelength encode information for plants/tell them about who is around?

Answer 16

open space has much higher quantity of light, much more blue green and red light

under a canopy = light transmitted through leaves (not absorbed) has much more far red light (lower R:FR ratio)

plant pigments like chlorophyll and carotenoids result in preferential absorption of blue and red light
Specifically more red light than far red light
So full sun = high levels of blue, and high R:FR

Neighbours - plant can be in full sun but perceiving light reflected from the leaves of neighbouring plants (competitors) because the reflected light has a lower R:FR ratio

Takeaway - for plants it’s not just light or no light, and not just for photosynthesis - light encodes information

Question 17

phototropism - Charles and Francis Darwin proposed?

Cholotny and Went looked at?

Answer 17

Charles and Francis Darwin - wrote about differential growth in response to stimulus like light. Proposed the ‘influence’ moved from site of perception to site of growth

Cholotny and Went - looked at asymmetrical distribution of auxin

Question 18

explain Winslow Briggs’ experiment to do with auxins

Answer 18

Looked at oat coleoptiles as a model for phototropism

Germinated seedlings in dark, which elongated (skotomorphogenesis)

Put foil caps on some of their tips (apex)
Added blue light from one direction
Ones with the caps didn’t show phototropism, confirming Darwin’s observations that response requires the shoot apex

Dissected apex were each placed on an agar block

Had a Control grown in the dark, then some bisected by cover slip (to prevent auxin in the agar block form diffusing uniformly)
Auxin found to accumulate in unlit side on bisected blocks

Question 19

Researchers used biochemistry to extract proteins from the stem region expected to initiate phototropism. what did they find?

Answer 19

a mystery protein

Phosphorylation assays identified a:
120kDa protein in pea membrane

Phosphorylated specifically in response to blue light

Followed same dynamics as phototropism

Found in numerous plants

BUT - couldn’t identify the protein - no genomics data, limited mass spec etc…

Question 20

with more tech available, Briggs performed some mutant screens on Arabidopsis - what did he find?

Answer 20

Germinated in dark, then exposed to unidirectional blue light, and identified the ‘non-phototrophic mutants’

Performed compensation to see if mutants were the same

NOTE - one of these mutants, NPH1, lacked the mystery phosphorylated membrane protein - so is most likely the gene for this ‘photoreceptor for blue light that mediates phototropism’

Question 21

how was NPH1 - the mystery protein - finally identified?

Answer 21

Really long and laborious process at the time, used different ecotypes and comparison of polymorphisms to locate NPH1 to Chr 3

Screened the Yeast artificial chromosome library with PCR markers and narrowed the gene down to a 7kb fragment

Complemented NPH1 mutant with this fragment – the mutant no longer showed the mutant phenotype so the fragment must contain the NPH1 gene

Used this to screen a cDNA library and identified a 3.3kb transcript the roughly expected size for the 120kDa protein

Question 22

explain NPH1’s domains and how it works

Answer 22

it is a blue/UV-A photoreceptor that regulates phototropism

note - a photoreceptor also requires a chromophore to perceive light

LOV domains - where the chromophore FMN (flavin mononucleotide) binds

LOV 2 - regulates the serine/threonine kinase domain (so that’s three key parts)

In blue light - the FMN is covalently bound to the LOV domains, and LOV2 stops its inhibition of the STK domain, so it can undergo autophosphorylation, and then phosphorylate some other things…

No blue light - FMN is non-covalently associated to the LOV domains, allowing LOV2 to inhibit the STK domain

Question 23

what is NPH3/what experiment showed this (briefly)?

Answer 23

This is a protein also required for phototropism identified in Briggs mutant screen

Rice experiment showed NPH3 is required for mediation of auxin gradient:
used radioactive auxin on WT and NPH3 mutant, the mutant had no auxin gradient in unidirectional blue light (while the WT did)

Question 24

specifically, what did use of GFP show about NPH3 and its role in the auxin gradient?

Answer 24

NPH3 + GFP showed localisation at the plasma membrane IN THE DARK
NPH3 was seen to be internalised IN LIGHT
NPH3 was internalised more on the light side than shaded (opposite to auxin) in UNIDIRECTIONAL LIGHT

Question 25

give a summary of what happens in blue light vs out of it, with the two proteins discussed (NPH1 and NPH3)

Answer 25

No light - Phot1 (NPH1) inactive, and NPH3 is membrane bound

Unidirectional light - Phot1 or NPH1 is active, auto-phosphorylates itself, phosphorylates NPH3, NPH3 is internalised on the side where there IS light

BUT - don’t really know how NPH3 causes auxin to accumulate on the opposite side…

Question 26

how does NPH3 cause auxin to accumulate on one side?

Answer 26

PIN proteins -

required for the movement of auxin through tissues, they are efflux carriers

while auxin can be considered a morphogen, it cannot freely diffuse and requires these pins for transport

They are localised to the membrane
Their distribution changes from being even in the dark, to being found more on the shaded side in unidirectional light

Question 27

just a bit more detail on PIN proteins and how they work?

Answer 27

localised to the plasma membrane and in a number of different circumstances, their localisation and polarity within cells has been shown to direct auxin movement and accumulation

So, for example, it is possible for all the PIN proteins to localise to the apical end of cells and if cells are in a file (one on top of each other) then this will lead to auxin being transported upwards

Question 28

PIN proteins - what are PIN mutants like?

Answer 28

PIN mutants were non-phototrophic but when three of them were not working (not the single or double mutants, suggesting some redundancy)

Question 29

give a summary now, of light and phototropism and the proteins involved

Answer 29

Phototropism involves stem bending towards light

It is mediated by Phototropin blue light receptors (NPH1)

Blue light activates PHOTs (NPH1) and in stems leads to NPH3 phosphorylation

Gradient of PHOT/NPH3 across the stem is inversely related to auxin distribution

Auxin redistribution requires PIN efflux carriers