Practical Value of Plant Studies: Plants can be Green Machines
Plants are a source of biofuel.
Derived from recently living biomass:
New plants with high biomass yield:
Switchgrass (prairie grass)
2. Value of Plant Genetic Studies for Basic Biology
1. For comparison
2. As additional examples
3. Because they are part of the natural world
a. Plants share a common eukaryotic ancestor with animals. Examples: Chromatin Cytoskeleton Golgi, ER, usual organelles Gene expression components Ga = giga-annum = billion years From Meyerowitz, 2002
C. Arabidopsis thaliana : A model system for flowering plants
1. Life cycle
Small plant, easy to grow
High fecundity (10,000 seed/individual)
Self and cross-fertilization
125 Mb, smallest known in plant kingdom
Little repetitive DNA
RFLP map between ecotypes
Arabidopsis is a member of the mustard ( Brassicaceae ) family, which includes cultivated species such as cabbage and radish. Meyerowitz. Ann. Rev. Genet. 21 : 93-111(1987)
1. Arabidopsis Life Cycle Life cycle of higher plants. (A) The dominant diploid generation (B) flowers (C) male and the female reproductive structures (anthers and siliques) (D) Gametes produced by meiosis: pollen and ovule (E) Fusion of pollen and ovule to form new diploid generation (embryo).
Haploid generation is multicellular
No dedicated germline
1. Two Features of Plant Development Relevant to Genetic Analyses
Gametes in plants are formed by a separate multicellular haploid generation called the gametophyte.
Multiple rounds of division in the haploid phase.
Implication for essential genes.
Plants have no dedicated germline. Instead, cells giving rise to the germline develop de novo from the somatic tissues.
Implications for environmental inputs into the production of the germline.
2. The Arabidopsis genome (Nature, 408:796-815; 2000)
Arabidopsis Molecular Genetic Tools A. Agrobacterium-mediated plant transformation
Agrobacterium tumefaciens & crown gall tumor Agrobacterium is a genus of soil bacteria that infects wounded plants and leads to gall formation Galls = benign tumor that feeds the extracellular agrobacteria
Agrobacteria induce tumors by genetically engineering plant cells at the wounded site
Ti plasmid ~200 Kb LB RB Virulence genes LB RB T-DNA introduced into plant chromosomal DNA LB RB T-DNA plasmid for transformation Selectable marker T-DNA acts as an insertional mutagen
Agrobacterium-mediated plant transformation “Floral Dip” Selection T1 Solution of Agrobacterium harboring a T-DNA plasmid
Method of mutagenesis
GFP fusions, reporter genes, etc.
T-DNA insertion at random locations in the genome T-DNA plasmid Gene A Gene B Gene C Examples of possible insertions: LB Selection RB YFG *
II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process pi-1 D. Map and clone mutation Infer mechanism & Generate hypotheses Mutant phenotype Responsible gene
A. Floral Organ Development in Arabidopsis 4 sepals 4 petals 6 stamen Male gametes pollen 1 carpel Female gametes ovules
A. Arabidopsis Floral Organs are Arranged in Whorls
Four concentric whorls of organs
Stereotyped pattern of number and position.
ca st pe se
II. Forward genetics B. Generate a mutant population C. Identify interesting mutants A. Select a biological process (Flower development) pi-1 D. Map and clone mutation
Our example: visual screen for floral development mutants of Arabidopsis.
Deviations in floral organ number
Deviations in floral organ location
Arabidopsis Homeotic Mutants Homeotic mutations cause conversion from one organ to another. ca st pe se apetala1 (ap1) apetala2 (ap2) ca se ca se pistillata (pi) apetala 3 (ap3) agamous (ag) ca st st ca pe se pe
The ABC Model of Floral Development Coen and Meyerowitz, Nature 1991
Three classes of homeotic genes
A function (AP1, AP2)-->sepals, petals
B function (AP3, PI)-->petals, stamen
C function (AG)--stamen, carpels
ap3 or pi ca se ca se B-function genes are required for the production of petals and stamens A C B
ap2 A function genes are required for the production of sepals and petals A C B ca st st ca
ag C-function genes are required for the production of stamen and carpels A C B pe se pe
can get additional alleles to corroborate data on previous EMS alleles
can study genes that are interesting because of evolutionary, biochemical, or expression data.
Can do a step-by-step analysis of the redundant functions of members of a gene family
Common outcome: no phenotype because gene is redundant or conditionally required.
Reverse genetics B. Generate mutant plants C. Evaluate mutant phenotype A. Identify gene or genes of interest pi-1 D. Identify the function of the genes Infer mechanism & Generate hypotheses Mutant phenotype Responsible gene
III. Reverse genetics: the Arabidopsis toolbox
Sequence-indexed T-DNA insertion/transposon lines
SALK, GABI-Kat, CHSL, RIKEN, SAIL, Wisconsin, etc.
Engineered Post-Transcriptional Gene Silencing.
Overexpression of wild-type or dominant-negative alleles.
Available SALK insertion lines for AP3 AP3 SIGnAL= S alk I nstitute G e n omic A nalysis L aboratory http://signal.salk.edu/ III. Reverse genetics: the Arabidopsis toolbox
Available SALK insertion lines for PI PI III. Reverse genetics: the Arabidopsis toolbox SIGnAL= S alk I nstitute G e n omic A nalysis L aboratory http://signal.salk.edu/
2. TILLING T argeted I nduced L ocal L esions IN G enomes
A high-throughput strategy for generating and isolating point mutations in your favorite gene
Exploits a nuclease that recognizes and digests heteroduplexes.
Mutations in genes that are not found in the insertional database.
Partial loss of function.
2. TILLING T argeted I nduced L ocal L esions IN G enomes EMS-mutagenized plant population * * CEL1 * * * * * * x x x Heat, anneal wild type and mutant versions Digest with CEL1 endonuclease digests heteroduplexes ONLY * * * * * * * * Pooled DNA from individual plants in 96 well plates PCR amplify your gene with fluorescently tagged primers from DNA in each pool Pool #104
Nature Biotechnology 18 : 455-457 (2000). Run on gel Screen individual samples, sequence TILLING T argeted I nduced L ocal L esions IN G enomes pools
Reverse genetics gave additional insight into floral development
We know MADS-box genes are required for floral development:
But they are not sufficient.
What are the missing factors?
The SEPELLATAS : A fourth class of floral organ identity genes From Current Opinion in Genetics and Development 2001 11 : 449
SEP1 identified as an AP3-interacting protein in the Y2H.
SEP1,2,3,4 are all highly similar MADS-box proteins.
The SEPALLATA genes are required for floral organ formation Wild type sep1sep2sep3sep4 Ditta, et al. Current Biology 14 :1935 (2004).
Turning Leaves into Petals Wild type Expression of AP1, AP3, PI, and SEP is sufficient to convert seedling leaves into petals. Pelaz, et al. Current Biology 11 : 182-184 (2001)