plant development and environmental regulation of development Flashcards
plants, like Arabidopsis, show polarity super early - how?
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
what are some restrictions of plant cells, that animal cells don’t have, when it comes to development?
no cell movement, rigid cell walls, shape is determined by cell division plane and cell expansion
in plant embryogenesis, why is the embryo proper called ‘1-cell’, but looks like 2?
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
using Arabidopsis as our model for dichotomous plants (meaning two cots at the heart stage), what are the organs of a flower?
Four organs, arranged in concentric ‘whorls’
All organs derived from an inflorescence meristem
Outer to inner - sepals, petals, stamen, carpals
what are three mutants affecting flower organs and their phenotypes?
what’s the first question then asked about these mutants?
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’
explain how AP1, PI and AG can explain organ expression of a flower
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
what is the ABC model?
important detail?
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
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?
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
what are the A, B and C genes?
Homeotic genes
MADS transcription factors (not homeodomain TFs)
Have a 50-60 Aa DNA binding domain
what is the ABCE model?
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
no B gene?
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)
no C gene?
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
why is environmental regulation of development studied in plants more than animals?
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
light as a language - three ways?
Photoperiod - day length
Quantity - the amount of light reaching a surface
Quality - the balance of different wavelengths
bit of background on light - why is the sky blue, clouds white, sunrise/sunset?
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)
how can light wavelength encode information for plants/tell them about who is around?
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
phototropism - Charles and Francis Darwin proposed?
Cholotny and Went looked at?
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
explain Winslow Briggs’ experiment to do with auxins
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
Researchers used biochemistry to extract proteins from the stem region expected to initiate phototropism. what did they find?
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…
with more tech available, Briggs performed some mutant screens on Arabidopsis - what did he find?
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’
how was NPH1 - the mystery protein - finally identified?
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
explain NPH1’s domains and how it works
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
what is NPH3/what experiment showed this (briefly)?
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)
specifically, what did use of GFP show about NPH3 and its role in the auxin gradient?
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
give a summary of what happens in blue light vs out of it, with the two proteins discussed (NPH1 and NPH3)
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…
how does NPH3 cause auxin to accumulate on one side?
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
just a bit more detail on PIN proteins and how they work?
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
PIN proteins - what are PIN mutants like?
PIN mutants were non-phototrophic but when three of them were not working (not the single or double mutants, suggesting some redundancy)
give a summary now, of light and phototropism and the proteins involved
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
