AGR2451 Lecture 5 - Notes (M. Raizada)•complete last week’s lecture•this lecture’s handout at the front•please pick up a questionnaire at the front•No Reading for this lecture:•Did you review your notes on Tuesday night???!!!-------------------------------------------------------------------Lecture 5 - Change I- “New Plant Traits from DNA Mutations”

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time (million years)

Moss

Arabidopsis

Corn

Fungi

Nematode

Arthropod

Fishes

Amphibians

Rodents

Cambrian

Precambrian

Animals Fungi Plants

Evolutionary Divergence of the Major Eukaryotic Model Systems

*Fossil evidence (often much later than molecular clock data)*Housekeeping/conserved nuclear or organellar genes*Micro and macro DNA changes (introns, inversions, duplications)

summarized from Proc.R.Soc.Lond.B.(1999) 266: 163-171 Nature (1997) 389: 33-39

Primates

Corn Arabidopsis

Land ecosystems, human civilization

Model Research SpeciesFor Plant Geneticists

Change: Evolution of Land Plant Phenotypes (see Labs)•Plants, unlike animals came onto land only once -- all land plants related•there are >250,00 species of land plants, most flowering plants

Bryophytes(eg. moss -9500 sp.)

Seedless vascular(eg. ferns - 11,000 sp.)

FreshwaterGreen Algae

Gymnosperms(720 sp. - eg. conifers)

Vasculartissue (sugarwater, etc.)

-Seed-

Onto dry land;secondarymetabolite

Flowering Dicots(170,000 sp. - eg. Arabidopsis,tomato, potato, peaches, canola)

Flowering Monocots(65,000 sp. - eg. the grasses:corn, rice, wheat, barley, rye)

>450 mya

130-200mya?Fruits,

StomatesSurface wax

Slide 5.2

Roots

Wind pollen

Slide 1Slide 5.3

1. By mutating ancestral genes, single-celled eukaryotes have created multicellular organisms with novel, divergent traits. However, all organisms share a common set of genes. Why??

Functions of genes in Arabidopsis (weed, Mustard)-Transcription 16.9% 3,018 genes-Protein synthesis 4.1% 730

-Protein fate (modifications, 9.9% 1,766Folding, compartment targeting)

-Signalling 10.4% 1,855(incl coordination of multiple cell types)

-Intracellular transport 8.3% 1,472(between compartments)-Metabolism 22.5% 4,009-Plant defence 11.5% 2,055-Growth 11.7% 2,079-Transport 4.8% 849

----------Source: TAGI (2000) Nature 408, 796-814 assigned 17,833

total 25,498 Why?? Because all cells must carry out basic functionssuch as transcription, protein synthesis and making plasma membranes. These are called “housekeeping genes”.

Slide 5.4

• Despite dramatic phenotype changes, a key conceptis that all organisms are remarkably related at the molecular level in the protein coding regions:•this may be the greatest discovery in Biology in the last 50 years!!eg. >30% of human genes may have a functional counterpart in yeast (fungus) after 1.4 billion years of evolution•Obvious related proteins between species pairs:Human -Fruit fly 61%Human-Worm 43%Human-Yeast (fungus) 46%•~52% of Arabidopsis (plant) genes have a recognizable DNA/protein sequence to genesof fruit flies, worms, fungus and humans

Source: IHGSC (2001) Nature 409, 860-921•out of 289 human disease genes, 48% were signficantly related to Arabidopsis genes!! •17 human disease genes are more similar to Arabidopsis genes than yeast, fruit fly or worm eg. Hereditary deafness, MYO15

Cystic fibrosis, ABCC7Source: TAGI (2000) Nature 408, 796-814

•The genetic codes of different plants are very related to one another.

Why is relatedness advantageous to research in agriculture?

Conservation of DNA (Genotype) During Evolution

•sometimes, DNA/amino acid sequences have diverged, but when the 3-dimensional shapes of proteins from distantorganisms are compared, they have the same shape and perform a related function•natural selection selects for protein shape and function regardless of the encoding DNA sequence•not only are DNA/protein sequences related, but the linearorder of genes may be shared between more closely related species (=chromosomal synteny): demoeg.mouse-cat-humaneg. rice-corn-wheat-barley-oat-sorghum (cereals)eg. tomato-potato-tobacco-canola-Arabidopsis•this suggests that during evolution, not only genes,but entire chromosomal segments were conserved:

Slide 5.5

Moore, Devos, Wang and Gale (1995)Current Biology 5, 737-739Current Biology Publishing Group, (UK)

Grass synteny chromosome map

Slide 5.6

2. If proteins are so highly conserved across species, then why are there such dramatic differences between specieseven closely-related species (humans to chimpanzees or turfgrass to corn)?

Asked another way, are all mutations equal in their effect?

To breed each of the following new traits, in what type of geneand location within that gene should mutations be selected for?

-to recognize a new pathogen?

-to make a leaf bigger in size?

-to create a plant with 5 fewer leaves?

-to cause 1000 new genes (responsible for drought tolerance)to be induced by a new stimulus such as heat?

-to change the amount of starch in a seed?

-to add a sugar group (-OH) onto a plant toxin to detoxify itfor human consumption?

Slide 5.7

To answer the previous questions posed:•Though proteins and thus DNA coding regions may beconserved, the regulatory regions are not. Small changesin regulatory regions can lead to dramatic changes in phenotype quickly. This is important in plant breeding. •Small changes in signalling molecules (transcriptionfactor binding sites, receptor-ligand recognition, hormonedose) can also lead to dramatic changes in phenotype.

Case Study: Maize evolved from its wild relative, Teosinte. The two plants can interbreed (same species), yet their shootarchitecture is dramatically different. Mutations in only 1major gene, Tb1, is responsible for this change.

Slide 5.8

Teosinte vs. Maize

QuickTime™ and aGIF decompressor

are needed to see this picture.

Tb1 gene

TB1 is a signalling protein, which regulates the number ofvegetative branches produced by a plant. Comparing theTb1 gene between teosinte and maize,most of the mutationsare in the regulatory region (as well as in the intron), notthe exons. Changes in the regulation and dose of the mRNAlikely explain the differences in plant architecture.

Indigenous people in Mexico selected for mutations in theregulatory region of Tb1 over a period of >300 years.

In addition to mutations in the regulatory regions, small amino acid changes at the binding sites of proteins (eg. enzyme-substrate binding site,or transcription factor binding site) can lead to dramatic changes in phenotype.

Slide 1

From Biology of Plants p.164P. Raven, R. Evert and S.EichhornWorth Publishers, New York, 1992

Slide 5.9

3. Do mutations happen randomly or are theytargetted to certain genes or certain parts of genes?

In the case of Tb1, if mutations occur randomly,why are there so few mutations between Teosinte andmodern maize in the exons of Tb1?

Natural Selection is the “filter” that the environment places on a phenotype, thus selectingfor or against mutationsin the genotype. demo

example-Dubautia (sunflower family) includes28 species in Hawaii and California-Rainfall diversity 40cm-1230 cm/year-Result: natural selection on leaf size-Is natural selection active or passive?

Most mutations have no effect (neutral) and hencethey simply build-up over generations (eg. in introns and in the DNA between genes on a chromosome)

4. Natural Selection vs. Human Selection•Natural selection took place over 1.4-1.6 billion years for plants in complex ecosystems, with competition from predators, struggle over resources with other plants. Plants were not selected for to serve humans, but rather to deter animals oruse them for seed dispersal. •Human selection on plants has been the exact opposite: It has taken place only for >10,000 years, for the benefit of the human herbivore, in a monoculture ecosystemwith much less competition. What are the consequences of these differences?

Slide 5.10

From Raven

•Mutations selected by human selection by farmers are a much more valuable resource for crop breeding than random mutations,or arguably, mutations selected by natural selection. Hundreds of thousands of these varieties exist in seedbanks around the world and are used in breeding programs to introduce new traits. •Many of these farmer-selected mutations are likely inregulatory regions of genes, signalling proteins and enzyme binding sites.

InternationalSeed Banks

•novel alleles causeddrought tolerance,cold resistance, disease resistance,pest tolerance

The Seedbank at The International Maize andWheat Improvement Center (CIMMYT), Mexico

Raizada

Slide 5.11

Slide 5.12

Walbot and Petrov (2001) PNAS 98, 8163-8164National Academy Sci. Press, USA, Washington, DC

5. What causes the (random) DNA mutations?

i) Point mutations, short insertions and deletions. eg. G to A. These are caused by mistakes during DNA replication.

ii). Parasitic DNA -- DNA parasites that duplicate themselves and “jump” into genes and between genes (called transposons, retroelements, or mobile elements)

•ancient jumping genes may have given rise to introns•highly repeated blocks of mobile elements are calledheterochromatin (gene-rich blocks = euchromatin)•examples of mobile elements in humans:SINEs 1.6 million copiesLINEs 868,000 copies Source IHGSC (2001) Nature 409, 860-921

•in humans and corn, 80-90% of genome consists ofmobile element parasites (“junk DNA”)

•mobile elements can carry new regulatory information or jump into gene switches (very important in plant evolution), and hence havecreated new alleles for plant evolution

gene gene gene

Slide 5.13

Consequences of Parasitic DNA (Mobile Genes)

•dilemma: corn, rice, wheat have highly related genes,similar # of genes (50-60,000) but the total amount of DNA in their genomes is very different:Rice 450 million base pairsCorn 2,600 million bpWheat 12,200 million bp (all haploid)

Why??•these genomes vary in the amount of “junk DNA”they have due to a high copy number DNA parasites•therefore, mobile DNA is responsible for genome expansion by creating blocks intergenic “junk DNA” blocks. **DNA quantity does not correlate with complexity in multicellular organisms.**Example of a corn jumping gene jumping in and out:demo

E x c i s i o n

( R e v e r s i o n )

T r a n s p o s o n

I n s e r t i o n

P i g m e n t a t i o n G e n e

Source: M.Raizada

Slide 5.14

Lecture 5 - Concept Summary1. Proteins are conserved across evolution, especially“housekeeping proteins”. Most important concept!!2. Even between non-housekeeping genes, the proteinsthey encode maintain their 3-D structures (only 1000unique folds).

3. Though proteins and thus DNA coding regions may beconserved, the regulatory regions are not. Small changesin regulatory regions can lead to dramatic changes in phenotype quickly. This is important in plant breeding. 4. Small changes in signalling molecules (transcriptionfactor binding sites, receptor-ligand recognition, hormonedose) can also lead to dramatic changes in phenotype.

5. Small amino acid changes at the binding sites of proteins (eg. enzyme-substrate binding site,or transcription factor binding site) can lead to dramatic changes in phenotype.

6. DNA mutations occur randomly. Mutations with no effect can accumulate. Mutations with phenotypic effectscan be selected by natural selection or human (breeding) selection and these new mutations can spread through thepopulation.

7. Two mechanisms responsible for creating DNA mutationsare spontaneous nucleotide “point mutations” and the insertion and excision of parasitic mobile DNAelements (“jumping genes”)