Arabidopsis Molecular Genetics HORT 301 – Plant Physiology

October 31, 2007Reading 1 (Alonso and Ecker (2006) Nature Reviews Genetics

Reading 2 (Page and Grossniklaus (2002) Nature Reviews Genetics

Function of all plant genes

Assumption – majority of genes in plants have conserved function, including those in crops

Lecture outline:

Genetic terminology

Arabidopsis thaliana, the current plant genetic model system

Resources for gene function determination

Phenotypic selection or screening of a T-DNA mutant population – forward genetics, identification of the mutated gene

Candidate gene based on predictions of function (phenotype) – reverse genetics

Terminology

Genetics – study of heredity/inheritance and function of genetic material

Gene – individual functional unit of the DNA that includes the promoter and encodes the messenger mRNA that is translated into a protein, i.e. locus

Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism

Molecular genetics – application of molecular biology to genetics, i.e. gene identification by DNA structure, genetic engineering

Genotype (genes) to phenotype (appearance and function)

Inheritance – single gene dominant and recessive phenotypes

Griffiths AF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM. 1996. An Introduction to Genetic Analysis. 6th ed. W.H. Freeman & Company

Yellow – 416 (3)Green – 130 (1)

Y (dominant) and y (recessive) are alleles

Allele – alternative state of a gene that may be different in each of the two chromosomes of the pair, dominant (Y) or recessive (y) allele

Mutation – change in DNA structure of a gene, alteration in gene function

WT siz1-2 siz1-3

Plant breeders improve crops by transferring “mutations” that confer better phenotypes/traits!!!!

See the maize example

Arabidopsis thaliana (L.) Heynh, plant genetic model – (Brassicaceae, mustard or crucifer family) common name Arabidopsis, rosette-type plant

25.1 (A) The shoot apical meristem in Arabidopsis thaliana; (B) An Arabidopsis plant

Morphological, developmental and molecular genetic attributes of Arabidopsis that make this plant a genetic model:

Small size – numerous plants can be grown in a small area

Short life cycle – six to eight weeks, 6 to 8 generations per year

Produces numerous seed – several thousand seeds per plant

Diploid (2n) – n = 5 (chromosomes)

Self-fertile – amenable to classic genetic manipulation by self or cross pollination

Natural variation – better alleles for certain traits in other accessions or ecotypes

Transformable using Agrobacterium tumefaciens – foreign DNA is easily transferred into the genome (complete set of genes and accompanying DNA)

Genome size is relatively small - ~120 x 106 bp, 26,819 genes encode proteins, 31,762 total including genes that encode miRNA, pseudogenes, and transposable elements, etc. http://www.arabidopsis.org/portals/genAnnotation/genome_snapshot.jsp

Genome sequence information is available, also cDNA sequence, http://signal.salk.edu/cgi-bin/tdnaexpress

Bioinformatic information, http://signal.salk.edu/, http://mips.gsf.de/proj/plant/jsf/index.jsp

mRNA expression data, https://www.genevestigator.ethz.ch/at/

Mutations for nearly every gene – mutants are available, http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm

Genetic resources for gene function identification - T-DNA insertional tagging mutagenesis - Agrobacterium-mediated transformation

Agrobacterium tumefaciens – crown gall disease, bacterial pathogen of plants that transfers DNA (T-DNA) into the plant genome during the infection process

Tumor cells produce carbon and nitrogen sources for use by the bacteria

Agrobacterium infection and tumor development

“Disarmed” Agrobacterium strains are used for genetic manipulation (engineering) of plants – bacteria are no longer pathogenic but are still capable of T-DNA transfer

T-DNA is inherited as a single dominant gene (locus)

T-DNA binary vector (plasmid) composition, e.g., pSKI015 - right border (RB) and left border (LB)

DNA between the borders is inserted into the plant genome (DNA), selectable marker gene

Selectable marker gene – e.g. herbicide resistance gene to “select” transformed plants

Insertions are random, 1.5 insertions per event

4X 35ST-DNA Vector – *pSKI015

Transformation

Mutant Plants

Plant Genomic DNA T - DNA

or

Disruption

Activation

LB 3’-ocs-bar-mas-5’ OriC RB

Agrobacterium T-DNA insertional (tagging) mutagenesis

Activation sequence – e.g., 4X 35S that can activate expression of a native gene depending on the insertion position

Primary interest is to alter the function (cause a mutation) of every gene by T-DNA insertional mutatgenesis

It is estimated that ~300,000 random insertions will “saturate the genome”, a mutation in each gene, a population of 300,000 plants

Process is referred to as “tagging”

“Tag” – insertion of the T-DNA “tags” the region in the genome because the T-DNA sequence can be located in the genome because of sequence

Identification of the flanking sequence

Floral transformation of Arabidopsis

Each seed that is transformed has a unique mutation

Transformation procedure - inflorescences are dipped into a solution containing Agrobacterium

Plants are grown in the greenhouse and seeds are collected

Selection of transformed plants – typically based on resistance to a toxic agent (transgene expression results in detoxification), in this instance the herbicide bialaphos

Population is maintained through seed, i.e. inherited

Generation of T-DNA tagged population

Herbicide selection of transformantsPropagation and collection of

seed

Phenotypic screening of the T-DNA mutant population – forward genetics

Identify Salt Responsive Mutants in a T-DNA Insertion Population

T1 GenerationSelect Transformed Plants (Heterozygous, BialaphosR, ~0.2%)

T2 Generation

Primary Screen for Mutants (NaCl sensitivity or insensitivity)

T3 GenerationConfirm Genetic Stability of Phenotype (Homozygous,

Recessive/Dominant)

Locate the “Tag” and Identify the Flanking Genomic Sequence

Root Development(C24)

Luciferase Imaging(C24RD29A::LUC)

Shoot Development(Col-0 sos3-1)

Stress StressStress

Isolation of NaCl mutants using different approaches

Identification of the mutation in Arabidopsis genome – location of the T-DNA insertion (tag) and determination of flanking sequence

Most mutations are recessive, both alleles of the locus are homozygous for the mutation

T -DNAL B R B

L B primer

random primer

random primer

P roc edure for loc ating T -DNA in the Arabidops is g enome

---AATAC AG T G C C G TG AC T T T G T T C T TAAC TC T G G G G C AT T T AT T C C AC TG T T G C ATC AG C TG A------

e.g. F lanking sequence : –importin protein (At5g49310)

R B primer

P C R product

S equence of T -DNA S equence of unknown gene

(3) B las t-s earc h of identified flanking s equenc e in Arabidops is g enome databas e (http://www.nc bi.nlm.nih.g ov/B L AS T /)

(1) T AIL -P C R

(2) S equenc ing

T -DNAL B R B

?

-importin

-importin gene – At5g49310

Nature (2000) 408:796-815

Arabidopsischromosomes

Reverse genetics – candidate gene identification based on prediction of function and phenotypic effects

Ca2+/CaM regulated transcription factor that regulates drought stress responses of plants, GTL family

R evers e genetics

(1) S elect a gene or genes (ex. Arabidopsis G T -element binding transcription factor family)

At1g76880 (GTL6)

At1g33240 (GTL1)

At1g76890 (GT-2)

At5g28300 (GTL2)

At5g03680 (GTL3/PTL)

At3g10000 (GTL4)

At5g47660 (GTL5)

At2g33550

At1g31310

At3g25990

At1g13450 (GT-1)

At5g01380 (GT-3a)

At2g38250 (GT-3b)

At5g63430

At3g10040

At1g76870

At1g2120099

100

100

100

100

66

56

54

20

75

79

54

46

97

G T -2 /G TL

G T -1

Phylogenetic dendogram

Mutant lines are available to the public

(2) Generate a collection of mutations in selected genes: Arabidopsis Biological Resource Center (ABRC) provides T- DNA insertion

lines in Arabidopsis genome to public ( www.arabidopsis.org )

Gene T-DNA mutations Insertion position

AtGT-2

(At1g76890)

gt2-1 (salk_014451) Exon

gt2-2 (salk_035328) 3’ UTR

AtGTL1

(At1g33240)

gtl1-1 (salk_005972) Exon

gtl1-2 (salk_044308) Intron

gtl1-3 (salk_101901) 5’ UTR

AtGTL2

(At5g28300)

gtl2-1 (salk_087253) Promoter

gtl2-2 (salk_020059) 3’ UTR

AtGTL3

(At5g03680)

gtl3-1 (salk_144638) Intron

gtl3-2 (salk_010031) Promoter

AtGTL4

(At3g10000)

gtl4-1 (salk_058993) Exon

gtl4-2 (salk_145331) Intron

AtGTL-5

(At5g47660)

gtl5-1 (salk_049268) Promoter

gtl5-2 (salk_078330) Intron

AtGTL-6

At1g76880

gtl6-1 (salk_106258) Exon

gtl6-2 (salk_072465) Exon

AtGTL1 (At1g33240)gtl1-2

L RLLB

L+LB L+LB L+LBL+ LB L+ LB L+ LB

no T-DNA

homozygous heterozygous

No T-DNA homozygous heterozygous

Select homozygous T - DNA insertion line

Evaluate phenotypes

(3) Tes t phenotypes of mutations (F or example : Drought s tres s)Col-0 (wild type) gtl1-2 gtl1-3

0

20

40

60

80

100

Col-0 gtl1-2 gtl1-3

Sur

viva

l (%

)

R evers e genetic approach for G T L 1 gene revealed that the function of G TL 1 is important for drought adaptation in Arabidops is