Arabidopsis – Ease of transformation epigenetics and

response to environment

- Purvi Gosrani

IntroductionArabidopsis (rockcress) is a genus in the family Brassicaceae

They are small flowering plants realted to cabbage and mustard

Arbidopsis thaliana – one of the model organisms used for studying plant biology and the first plant to have its entire genome sequenced

It has small generation time and short genome size

In addition, Arabidopsis has proven to be an ideal organism for studying plant developement at the molecular, cellular, organismal and ecological level

TransformationThe ability to move DNA into an organism and thereby alter its phenotype is central to both basic and applied molecular biology

Transformation is a simple task with escherichia coli or Saccharomyces cerevisiae, but is usually more difficult with multicellular eukaryotes and can be particularly challenging with some important plant species

However, for Arabidopsis, in planta transformation methods have been developed that are incredibly simple

Attempts to apply in planta transformation methods to other plant species have often failed

This may be due in part to a poor understanding of the mechanisms that umderlie the successful Arabidopsis transformation method

Transfer of genes encoding growth factors and bacterial nutrients

Agrobacterium transfers the T-DNA containing genes that encode the protiens involved in biosythensis of plant growth factors and bacterial nutrients

T-DNA does not contain any useful gene to be transferred

Therefore a foreign DNA can be inserted into the plant chromosome by putting the desired sequences between two T-DNA borders and using the Agrobacterium mediated system

Utilising this natural property of transformation many variants of Arabidopsis have been developed

Main advantage of T-DNA insertion is it does not require additional steps to stabilise the insert

Transposon TagggingTransposon tagging has become a powerful tool for isolating new genes

It was first recognized by McClintock

The first successful cloning of plant gene was achieved via the Ac/Ds transposon system

Unlike T-DNA, transposons can be excised from the disrupted gene in the presence of transposase

It results in functional revertants that can confirm the phenotypic cosequences of the mutations

Many important genes required for the growth and better functioning of maize and corn have been successfully transplanted in Arabidopsis and studied

Arabidopsis Transformation Without

Tissue CultureAgrobacterium was applied to Arabidopsis seeds, grew plants to maturity in the absence of any selection, then collected progeny seeds and germinated them n antibiotic – containing media to identify transformed plants

Successful rounds produced transformants at a high enough rate that thousands of transformed lines were produced in a matter of a few years

These “insertional mutagenesis” lines helped speed gene cloning by the Arabidopsis community

Transfer of resistance genes

Another method of transformation was by “vaccum infiltration”

Arabidopsis plants at early stages of flowering were uprooted and placed into a bell jar in a solution of Agrobacterium

A vacuum was applied and then released, causing air trapped within the plant to bubble off and be replaced with the Agrobacterium solution

Plants were transplanted back to soil, grown to seed, and in the next generation stably transformed lines could be selectable using antibiotic or herbicide appropritae for the selectable marker gene

(A) Arabidopsis, plants are grown to a stage when they have just started to flower

(B) plants are dipped briefly in a suspension of Agrobacterium, Suc, and surfactant

(C) plants are maintanied for a few more weeks until mature and then progeny seeds are harvested

(D) and seeds are germinated on selective medium (e.g. Containing kanamycin) to identify successfully transformed progeny

Transcriptome Changes for Arabidopsis in Response to Salt, Osmotic, and Cold Stress

To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants

They were subjected to stress treatments of 4 degree celsius, 100 mM NaCl

RNA samples were collected separately from leaves and roots after 3- and 27- h stress treatments

After the complete test it suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions

The majority of changes were stimulus specific

At the 3- hr time point less than 5% of genes were showing stress response

A stress response was observed for 68% of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to “anticipate” predictable stresses such as cold nights

Environmental Regulation of Lateral Root Initiation in Arabidopsis

Plant morphology is dramatically influenced by environment signals

The growth and develpoment of the root system is an excellent example of this development plasticity

The development of lateral roots is may be due to transduction pathway that interprets complex environment conditions and makes the “decision” to form a lateral root at a particular time and space

When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ration, lateral root initiation is dramaticallly repressed

Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation

A mutant lateral root initiation 1 (lin 1) was isolated that overcomes the repressive conditions

This mutant produces a highly branhed root system on media with high sucrose to nitrogen ratios