1. Prospecting for Genes that Fueled the Green Revolution
Photo: Taiz and Zeiger, PLANT PHYSIOLOGY 5e

2. 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

4. Select a sequence and name your project:

5. Select genomes to search:

6. Click Run, when complete, the R stops blinking; Click Alignment View next:

7. Alignment viewer reveals a tree with the alignment behind it.

8. Note that each plant includes a set of these genes:
they are members of a gene family

9. Calculate a new tree using Neighbour Joining BLOSUM62

10. Do patterns emerge here?
How many major groups are there?

11. How many closely related genes does each species appear to have?
These genes are members of a gene family.

12. 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?

13. 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?

14. Monocots and dicots are the two major branches of flowering plants
Oryza
Zea
Dicots
Arabidopsis
Medicago

15. Do the monocots and dicots group separately?
Gene duplication event
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?

16. Oryza (rice)
Avena (oats)
Hordeum (barley)
Triticum (wheat)
Setaria (foxtail millet)
Pennisetum (pearl millet)
Sorghum
Zea (maize)
Arabidopsis
Brachypodium
Glycine max (soy)
Dicots 46 25
Monocots
50-70 13 14 28 9
Present 20 40 60
Pulled from iplant intro presentation
Time (million years)
- Genome duplication event
Based on the distribution of monocots and dicots in our tree could an ancestral gene duplication explain the tree that we see? 16

17. 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

18. 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

19. What are Gibberellins (GAs)?
A class of plant hormones
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
GAI
gai
Pulled from iplant green revolution presentation
Images: GreenRevolution_16.ppt

20. DELLA proteins include several key domains:
Figure from: Taiz and Zeiger, PLANT PHYSIOLOGY 5e

21. When GA is not present DELLA proteins bind transcription factors (including PIF) to repress transcription: X
PIF
PIF X
No transcriptional activation
No free PIF
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e, FIG

22. With GA: GA and DELLA proteins bind GID1 releasing PIF, free PIF regulates transcription:
Regulate genes
PIF
PIF
+ GA: Transcription factors released
+ GA
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e, FIG

23. Binding of DELLA proteins to GID1 with GA causes degradation of the DELLA proteins

24. Binds transcription factors including PIF
Binds GID1
Binds transcription factors including PIF
Mutations in different domains have different affects
Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e

25. 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

26. 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

27. 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:
©2011 by American Society of Plant Biologists

28. 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

31. 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?

32. Another useful tool we have available calculates a pairwise alignment
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.

33. 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?

34. How might mutations in the GRAS domain affect growth? Why?
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?

35. 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)

36. 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!

37. Binds transcription factors including PIF to repress transcription
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
If unable to bind GID1 DELLA proteins are not degraded in presence of GA—transcription factors always bound
C-terminal mutations often lead to tall phenotypes
Act as if GA is always present if transcription factors are always free

38. Binds transcription factors including PIF to repress transcription
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?

39. 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., Plant Physiol 157:
Dominant and Pleiotropic Effects of a GAI Gene in Wheat Results from a Lack of Interaction between DELLA and GID1