Download Modified from Taiz and Zeiger, PLANT

Prospecting for Genes that
Fueled the Green Revolution
Photo: Taiz and Zeiger, PLANT PHYSIOLOGY 5e
Learning Objectives
• Discover how changes in individual genes produce
phenotypic change
• Learn to apply bioinformatics tools to identify
related genes and investigate their evolutionary
relationships
• Understand that genes often are members of gene
families that arise through gene duplication
• Be able to apply sequence analyses to identify
mutations underlying specific phenotypes.
• Understand how selection for specific phenotypes
drove the Green Revolution
Select a sequence and name your project:
Select genomes to search:
Click Run, when complete, the R stops blinking;
Click Alignment View next:
Alignment viewer reveals a tree with the alignment behind it.
Note that each plant includes a set of these genes:
they are members of a gene family
Calculate a new tree using Neighbour Joining
BLOSUM62
Do patterns emerge here?
How many major groups are there?
• How many closely
related genes does
each species appear to
have?
• These genes are
members of a gene
family.
Return to the alignment:
• What is the major difference between the two groups of
sequences?
• These two groups are referred to as “DELLA” (purple
above) and “non-DELLA” proteins (pink above)…see why?
• Where do the DELLA
and non-DELLA
members of the gene
family fall on this
tree?
• What processes could allow gene families to come
about through evolution?
Monocots and dicots are the two
major branches of flowering
plants
• Dicots
– Arabidopsis
– Medicago
• Monocots
– Oryza
– Zea
• Do the monocots and dicots group separately?
• Given this, what is the most likely timing of the
major genetic changes that gave rise to this gene
family?
Glycine max (soy)
Dicots
Arabidopsis
Oryza (rice)
46
150-300
Avena (oats)
25
Brachypodium
Monocots
50-70
13
Hordeum (barley)
Triticum (wheat)
Setaria (foxtail millet)
14
Pennisetum (pearl millet)
28
Sorghum
9
Zea (maize)
60
40
20
Present
Time (million years)
- Genome duplication event
http://gfx.dnalc.org/files/evidence/Presentations/Intro_16.ppt
Based on the distribution of monocots and dicots in our tree
could an ancestral gene duplication explain the tree that we
see?
How can mutations in DELLA
proteins affect plant growth and
yield?
Let’s see if we can figure that out!
Photo:Taiz and Zeiger, PLANT PHYSIOLOGY 5e
What is the function of DELLA
and non-DELLA proteins?
• The DELLA proteins respond to gibberellins, a
class of plant hormones
• Gibberellic acid (GA) was one of the first found
• DELLA proteins transduce this signal using a
mechanism that shares features common to many
plant signal transduction pathways
• Mutations in these genes can cause GA
insensitivity—hence the name GAI genes
What are Gibberellins (GAs)?
•
•
•
•
A class of plant hormones
GAI
gai
Affect growth, cell elongation
Enhance seed germination, fruit production
Mutations in genes encoding proteins needed for GA
biosynthesis or sensing can cause dwarfism or unusually
tall plants.
• The reduced height and higher yield of Green Revolution
wheat and corn due to mutations in DELLA proteins that
function in GA sensing
Images: http://gfx.dnalc.org/files/evidence/Presentations/ GreenRevolution_16.ppt
DELLA proteins include several key domains:
Figure from: Taiz and Zeiger, PLANT PHYSIOLOGY 5e
When GA is not present DELLA proteins bind
transcription factors (including PIF) to repress
transcription:
PIF
X
No free PIF
PIF
X
No transcriptional
activation
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e, FIG. 20.19
With GA: GA and DELLA proteins bind GID1
releasing PIF, free PIF regulates transcription:
PIF
PIF
Regulate
genes
+ GA: Transcription
factors released
+ GA
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e, FIG. 20.19
Binding of DELLA proteins to GID1
with GA causes degradation of the
DELLA proteins
Binds GID1
Binds transcription
factors including PIF
Mutations in different domains have different affects
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e
Summary of GA and DELLA
protein interaction
• Without GA present
– DELLA proteins bind transcription factors inactivating
them
• With GA present
– DELLA proteins bind GID1 and are targeted for
degradation
– Released transcription factors regulate genes that
control stem elongation and affect seed production
Semi-dwarf grains: key phenotype for
increased yields
•
•
•
•
Resistant to “lodging”
Energy re-routed from growth to grain production
Mature faster, allowing multiple harvests per year
Turned developing countries into self-sufficient grain producers
Photo:Taiz and Zeiger, PLANT PHYSIOLOGY 5e
Alleles of DELLA proteins produce a range of dwarfism in wheat: the
genes are named Rht in wheat for Reduced Height
Pearce S et al. Plant Physiol 2011;157:1820-1831
©2011 by American Society of Plant Biologists
Identifying mutations in DELLA
proteins
• Next we will:
– Return to the Yellow Line
– Prospect for mutations in the GAI gene of
Arabidopsis
– Identify the location of the mutation within the
protein
– Discuss how these mutations can lead to the
phenotypic effects that are observed
•
•
•
•
Locate the mutant allele in the alignment.
Where is the sequence change (i.e. mutation)?
What phenotype would you predict to see with this mutation?
Why?
•Another useful tool we have available calculates a pairwise
alignment. Let’s do that here.
•Select the gai protein and a very similar Arabidopsis
sequence
•From the calculate menu, select pairwise alignment.
• The mutation—a deletion in this case--jumps right out in this
alignment.
• How many amino acids long is the deletion?
• Can we determine the exact length of the deletion in nucleotides
based on this information? If so, how long is it?
Modified from Taiz and Zeiger,
Plant Physiology, 5e
Binds GID1
Binds transcription factors
including PIF
To repress transcription
• Knowing these functional domains, what phenotype would you
predict for plants with the mutation that we have identified?
• How might mutations in the GRAS domain affect growth?
• Why?
Now run your own analysis to
locate a mutation
• Exit back out to the Yellow Line
• Attempt to identify the dwarfism mutation
gai D8-1 mutant in Zea mays (corn)
D8-1 mutation
• What phenotype could be expected as a result of this
mutation?
• Prepare a diagram that illustrates how this mutation can
lead to the predicted phenotype.
• Now, prepare a diagram (or use the one above) to
illustrate how this mutation could lead to the opposite
phenotype—this is a challenge!
Modified from Taiz and Zeiger,
Plant Physiology, 5e
Binds GID1
• N-terminal mutations often
lead to dwarf phenotypes
• If unable to bind GID1
DELLA proteins are not
degraded in presence of
GA—transcription factors
always bound
Binds transcription factors
including PIF to repress
transcription
• C-terminal mutations often
lead to tall phenotypes
• Act as if GA is always
present if transcription
factors are always free
Modified from Taiz and Zeiger,
Plant Physiology, 5e
Binds GID1
Binds transcription factors
including PIF to repress
transcription
• N-terminal mutations often lead to dwarf phenotypes
• What phenotype might you observe if the mutant
protein bound GID1 whether or not GA is present?
Want to learn more about DELLA proteins?
• A wide variety of mutations in DELLA proteins are known.
• How some of these cause the observed phenotype is
obvious…others are more challenging to understand.
• More interesting examples are discussed in:
Wu et al., 2011. Plant Physiol 157: 2120-2130
Dominant and Pleiotropic Effects of a GAI Gene in
Wheat Results from a Lack of Interaction between
DELLA and GID1