GhDREB Transcription Factors

Pants are easily affected by external conditions like temperature, light, water availability, etc. If these factors exceed their optimum range for the plant, then plants may undergo some stress conditions. Drought stress, salt stress, and temperature stress (may be hot or cold) are some major types of stresses that are normally faced by plants.

Plants introduced many adaptations in themselves to deal with these stress conditions. These adaptations start with stress stimulus and end with different gene expressions. The expression of genes under stress conditions is led by transcription factors. For example, DREB1 is a transcriptional factor that is induced in plants when they face low-temperature conditions and increases the tolerance level in plants against cold temperature stress. However, over-expression of it results in a decrease in the plant’s height and also delays the flowering in plants. Another transcriptional factor from the same family, DREB2 is induced in plants under dehydration stress conditions. A natural immune system against these kinds of stress conditions is also present in plants which is well regulated by the plant’s genes. It's very important to identify these genes that resist the stress conditions.

Cotton is an economical plant and plays a vital role in maintaining the economy of many countries. Cotton fiber is used in making currency notes, bandages, and swabs and it plays an essential role in the textile industry all over the world. But stress conditions like drought stress, hot and cold temperature stress, etc. affect the production of cotton fiber very severely.

One of the transcriptional factors that regulate the stress resistance genes and help to form mRNA through the process of transcription in cells is the DREB (dehydration-responsive element Binding) protein. It is a sub-family of ERF (Ethylene Responsive Factor). The DREB1 transcription factors were first discovered in Arabidopsis thiamine. This family of transcription factors enhances the tolerance of different plants against drought and salinity stress. For example, transcription factors GhDREB1 is induced in cotton plants under low-temperature stress while GhDREB2 is induced in cotton plants under drought stress conditions (Reference Paper). Both of the transcription factors belongs to the same family i.e. DREB proteins but are induced in plants under different stress stimulus. the DREB genes can be further divided into six different sub-groups (A1 - A6).

Here, we will discuss about GhDREB1 (transcription factor). it is induced in cotton plants under stress conditions which then leads the gene expression through the trancription (formation of mRNA from DNA) process. GhDREB1 is isolated from the cotyledons of cotton plants by a group of researchers which was led by Da-Peng Shan, Jin-Guang Huang, and their other team members. They published their research paper in 2007. They found that GhDREB1 helps cotton plants to survive in low-temperature conditions. However, this transcription factor is negatively regulated by gibberellins. Gibberellins is a hormone that helps in the seed germination of plants. It is a growth hormone that inhibits the effect of the GhDREB1 factor in cotton plants.

Isolation of GhDREB1 from the cotyledons of Cotton.

They took the ZM19 variety of cotton seeds.
Then they grow these seeds in a growth chamber.
The light intensity in the growth chamber is about 300 µmol m–2 s–1 and the day-night temperature is about 20-28℃.
When these young seedlings are about 20 days of age, they divide the samples of seedlings into two halves in numbers.
The first half seedlings were treated with a cold temperature of about 0℃ for a specific range of time.
The second half of the seedlings is cultured in the solution of NaCl, abscisic acid, or polyethylene glycol for a specific range of time.
Then they preserved the leaves, roots, and stem of these seedlings in a liquid nitrogen solution (i.e. under -80℃) for later use.
The mRNA was isolated from cotyledons of the leaves of the seedlings that were treated at 0℃. temperature stress.
They form the strands of cDNA from the mRNA.
Then they amplify the strands of cDNA by PCR (polymerase chain reaction).
The protocol used in PCR is the SMART (Switching Mechanism At 5' end of RNA Template) kit.
This kit allows us to make full-length strands of cDNA from small samples of mRNA without any RNA adaptor ligation.
Then they treated these double-stranded stands of cDNA with Sfill enzyme (a restriction endonuclease enzyme that identifies the sequence of DNA and breaks it at that site).
They separate and purify the cDNAs by using Chroma Spin-400.
It is a product of Clontech company which is a spin-based column purification system. This system separates and purifies the fragments of DNA.
Then they cloned the fragments of DNA that are longer than 500 bp by using Sfill digested dephosphorylated λTripIEx2 arms with T4 DNA ligase.

λTripIEx2 arms are the vectors that are used in the cloning of DNA fragments in bacteriophage cells.
These vectors are dephosphorylated to prevent the self ligation and promote the ligation of foreign DNA (i.e. cDNA).
The T4 DNA ligase is the enzyme that catalyzes the phosphodiester bonds between the nucleotides of DNA.

In this way, the cloning of cDNA by using Sfill digested dephosphorylated (λTripIEx2 arms with T4 DNA ligase) takes place, and a recombinant DNA is formed.

The recombinant DNA was then packaged in vitro by using Packagene.
Once the clones of cDNA were prepared, they used the differential hybridization method to identify the specific clones of recombinant DNA.
It is a useful technique to identify the genes that were expressed in different conditions and also genes of different cells of a tissue.
The differential hybridization was done on two different kinds of cDNA.

One cDNA is taken from a cotyledon which is placed under 0℃. for a specific time.
The second cDNA is taken from the cotyledons of normal or untreated cotton plants.

The plaques on the plate (with a diameter of 15cm) were transferred to the membrane, and the density of the plaques was 10,000.
It means 10,000 individuals of plaques on the plate (within 15cm diameter) were found. The term plaques refers to the region of small circular areas of bacterial growth that has a single type of virus or bacteriophage.
Prehybridization, hybridization, and washing were performed to identify the clones of the cDNA of cotyledons that were treated with 0℃.

Prehybridization means that the membrane is incubated in a solution to block any non-specific binding sites for the probe.
Hybridization means that the membrane is incubated with the probe so that it will bind only to the sequence of cDNA.
In washing membrane is washed to remove any unbound probe.

To get more accurate results (i.e. clones of cDNA of cotyledons treated with 0℃), two more rounds of plaque hybridization are performed.
Clones that were exclusively hybridized (cotyledons treated at 0℃) were selected.
Out of these clones, GhBRED1 is taken.

Results of their Experiment.

GhDREB1 factor can be induced under cold temperatures as it increases tolerance capacity under cold temperature stress conditions by regulating gene expression.
However, over-expression of it may decrease the flowering and also decrease the height of the plant.
It is negatively regulated by gibberellins.
However, stress hormones like abscisic acid (induced in ABA-independent pathways) did not play any role in the production of GhDREB1 factors.
Drought stress is also not involved in generating this transcription factor (Research Paper).
DREB genes can lead biological processes in plants like growth, development, and stress relief by regulating their targeting genes through transcription and post-transcriptional processes.

The expression of the DREB genes family is not only seen in cotton plants. Other plants also have DREB transcription factors in stress conditions that play a great role in maintaining a tolerance level in these plants. Some examples of these plants are (links are redirected to the research papers of the respected plants):

Wheat (TaDREB)
Sugarcane (SoDREB)
Mung bean (VrDREB)
Barley (HvDREB)
Tomato (SlDREB)
Potato (StDREB)
Banana (MaDREB)

The DREB gene family is a very diverse family as it performs different roles in plants under different conditions. It helps in gene regulation through the process of Transcription. We can observe the DREB gene expression by amplifying the complementary DNA (cDNA) in PCR (polymerase chain reaction) and then observing it on gel electrophoresis.