Prospecting for Genes that Fueled the Green Revolution

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

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 BLOSUM 62

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 Monocots Brachypodium 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 5 e

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/ Green. Revolution_16. ppt

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

When GA is not present DELLA proteins bind transcription factors (including PIF) to repress transcription: PIF X No free PIF X No transcriptional activation Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5 e, FIG. 20. 19

With GA: GA and DELLA proteins bind GID 1 releasing PIF, free PIF regulates transcription: PIF Regulate genes + GA: Transcription factors released + GA Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5 e, FIG. 20. 19

Binding of DELLA proteins to GID 1 with GA causes degradation of the DELLA proteins

Binds GID 1 Binds transcription factors including PIF Mutations in different domains have different affects Modified from Taiz and Zeiger, PLANT PHYSIOLOGY 5 e

Summary of GA and DELLA protein interaction • Without GA present – DELLA proteins bind transcription factors inactivating them • With GA present – DELLA proteins bind GID 1 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 5 e

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, 5 e Binds GID 1 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 D 8 -1 mutant in Zea mays (corn)

D 8 -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, 5 e Binds GID 1 • N-terminal mutations often lead to dwarf phenotypes • If unable to bind GID 1 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, 5 e Binds GID 1 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 GID 1 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 GID 1