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.

• 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?

Return to the alignment:

• 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• Monocots

– Oryza

– Zea

• Dicots– Arabidopsis

– Medicago

• 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?

50-70

46

28

25

13

14

9

150-300

Monocots

Dicots

Time (million years)Present204060

Oryza (rice)

Avena (oats)

Hordeum (barley)

Triticum (wheat)

Setaria (foxtail millet)

Pennisetum (pearl millet)

Sorghum

Zea (maize)

Arabidopsis

Brachypodium

Glycine max (soy)

- 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?

http://gfx.dnalc.org/files/evidence/Presentations/Intro_16.ppt

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

• 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

What are Gibberellins (GAs)?

GAI gai

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

Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5e, FIG. 20.19

When GA is not present DELLA proteins bind transcription factors (including PIF) to repress transcription:

No transcriptional activation

PIF PIFX

No free PIF

X

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:

+ GA

+ GA: Transcription factors released

PIF PIFRegulate genes

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?

Binds GID1 Binds transcription factors including PIFTo 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?

Modified from Taiz and Zeiger, Plant Physiology, 5e

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!

Binds GID1 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

• 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 GID1 Binds transcription factors including PIF to repress transcription

Modified from Taiz and Zeiger, Plant Physiology, 5e

• 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?

Wu et al., 2011. Plant Physiol 157: 2120-2130

Dominant and Pleiotropic Effects of a GAI Gene inWheat Results from a Lack of Interaction betweenDELLA and GID1

• How some of these cause the observed phenotype is obvious…others are more challenging to understand.

• More interesting examples are discussed in:

• A wide variety of mutations in DELLA proteins are known.

Want to learn more about DELLA proteins?