Plant Growth Question Page

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South City High School Student BB: 16th April 2pm - Can you please explain why................... 

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Plant Growth 2009 Questions Page

Thank you for the questions that came in during the seminar via wiki-chat and from our live audience.

These, along with answers from the seminar will be posted here on Monday 24th August

If you want to post new questions and for comments on the challenge, please go to the discussion page 



1.     KB and MH 20th August

What is the difference between quantitative Real Time (RT) PCR and reverse transcriptase PCR? Why can’t the reverse transcriptase PCR be used to show the expression of the gene?

Karine David - from the seminar

In Reverse Transcriptase PCR (RT PCR) mRNA is extracted from tissue (e.g. plant tissue) and used to synthesise complimentary DNA which is the template for the PCR.  Multiple copies of the target DNA are made from this in the PCR process.  When the PCR has finshed, the product is tested by running it through a gel electrophoresis.  If the target DNA has been copied in the PCR process, a band of DNA the size of the target will appear on the gel.  This simply tells us whether the gene is being expressed or not - it does not tell us anything about how much gene we have.

Real Time PCR uses exactly the same system of extraction of mRNA from tissue that is used to synthesise complimentary DNA which is them copied in the PCR process.  The difference is that in real time PCR fluorescent tags are attached to the double stranded DNA.  This means that as the PCR reaction proceeds, as new peices of double stranded DNA are synthesised, the stregth of the flurescent signal increases. This means that in "real time" you can see a curve forming which indicates now much of the target DNA is present. 


2.    The research has shown that when ABP1 gene is down-regulated (inactivated) plant growth is reduced. Does this mean that if the gene is up-regulated (overactive) plant growth will be increased? 

Karine David - from the seminar
When you over-express you can make a lot of the gene - but that does not mean that you will get a lot of the protein in the right place! It may mean that you get more protein, but it comes out in places that it should not be. That is what happened when we created a model where we could turn on over-expression of ABP1 in Tobacco. But we found out that if you them add a lot of auxin, you can increase cell expansion in the leaves. This is what we hope to be able to do with the apples. Interestingly when we increased the cell size of the leaves the leaf size remained the same. This means that we had fewer, bigger cells.
'While there are different forms of auxin, they will always trigger the classic growth responses - cell division, phototropism, gravitropism, increased lateral root development etc - but it depends on the concentration.  The effect will be different at different concentrations.


3.    Tauranga Girls' College RH and TG 20 August

Why did you use ethanol vapour to turn the APB1 gene on and off? Was it a chance discovery or was the transgenic gene specifically chosen as one that responded to ethanol?

Nathan Deed - PhD student in the Plant Sciences Group at SBS 25 August 
The ethanol does not actually turn off the gene for ABP1. What happens is that the ethanol turns on a small gene which makes a piece of an antibody to ABP1. Karine called it a mini-antibody.  This mini-antibody binds to ABP1 protein and inactivates it. There are other systems for inducing gene expresion (turning the gene on) but many are rather fiddly. Ethanol is readily available, cheap not toxic at the levels used and easy to apply to the plant. It was not a chance discovery, but it was known that there was a piece of DNA (a promoter) in a fungus that responded to ethanol and this was cloned and used in a plasmid together with the mini-antibody gene, then used to transform the plants.


4.    Onehunga High School (from wiki-chat)

You used ethanol to inactivate Auxin Binding Protein 1 in the GM plants.  Is the effect of ethanol on plant groth permanent or can the effect be reversed?

Karine David - from the seminar

This is a very good question. We have shown that it is reversible. If we expose the plant to ethanol and grow it for 2-3 days we get the reduced growth. If you take away the ethanol, this is reversed. The longest period of time we have tried reversal with is 4 days, but it would probably still reverse after a week.



5.      Sarah Hunter, Parnell College
Is the TIR1 receptor specific to one type of auxin or will it bind to any forms of aucin?


Karine David - from the seminar
.
It can bind to all auxins that could be found in the plant. We tend to find one main auxin - IAA or indole-acetic acid, in the plant. There are multiple varieties of auxins, however they all have a similar structure and are all weak acids. They all have a ring structure and an acidic component. They all have the ability to bind to TIR1.



6.      From wiki-chat

Is there any reason why you chose the plants that you used in the experiments and would you get the same results from all plants?

 

Karine David, from the seminar

We used Arabidopsis and Tobacco  plants in this research. These plants are used because they are a "model plant systems". The model plants are chosen because they are plants which are easy to grow in large numbers; they are plants that we can make into transgenic plants so that we can turn a gene or protein on or off; they are plants where we can easily follow genetic patterns and we know as much of the genome as possibly.  We would expect the results to be transferable to most other species.


7.     Waimea College - MJ - From wiki-chat

How does ethanol inhibit ABP1 in a transgenic plant but not in a normal plant?

Karine David, from the seminar

The transgenic plant has an extra gene inserted into it which codes for a mini-antibody. Ethanol is used to "induce" (or turn on) the expression of this antibody gene to make the antibody. This will only inactivate ABP1 in the transgenic plant because the wildtype (normal) plants do not have the antibody gene so when ethanol is added to the environment of the wild type plant it has no effect. The mini-antibody was chosen because it was found to inactivate ABP1 if it was present and activated in the plant.  In fact the mini-antibody gene is placed randomly in the genome - so where it is does not matter. because the antibody acts on the ABP1 protein rather than affecting the gene for ABP1. 



8.     Senior College - Live audience

What impact will the research that you are currently doing on apples have on us as consumers?

Karine David, from the seminar
It will take a long time to reach the consumer - but the process has started. This research will help us to identify how we can modify cell size in the plant and therefore improve the quality of fruit - and have a positive impact on the economy. NZ will not be growing transgenic plants. However the current transgenic research will be used to inform how to create improved fruit. Currently at Plant and Food Research is a method called cease-genetics is being explored. Rather than using agrobacterium t-DNA to create a transgenic plant, we try to find the same sequence of DNA that inactivates ABP1 from within the plant and add this to the genome in our target apple plants. This means that the plant we create is not transgenic, but we are still able to turn on or off the gene.




9.     Onehunga High School - from wiki-chat

Can you target auxins in some parts of the plant?  Can you make some parts of the plant grow and some not?

Karine David, from the seminar
Yes - it is all about manipulating gene expression where you want, and what you want, at the time that you want.

There are tissue specific promoters on the genome. (Remember the promoter is the sequence of DNA sitting before the gene that the transcription factor will bind to.  Once it is bound, the process of transcription of the gene will start (i.e. making mRNA and then translation into a protein)). So you can have your gene expression (and therefore the growth effect) in a tissue that you want and you can even control whether it is turned on or not using specific chemicals. 

10.     Auxins sometimes make plants grow but sometimes kill plants.  How does that work?

The "killing growth effect" of auxins is a bit different. The effect of auxin is dependent on the tissue, the species, and the concentration of the hormone. When auxin is being used as a weed-killer, we are using the fact that auxin has a different effect on monocotyledons (e.g wheat or barley etc) and dicotyledons (e.g. dandelion), and that the effect on these two different plant groups are different at different concentrations. When we spray a crop like wheat with auxin at the concentrations used for weed killers, the monocotyledon (wheat, corn etc) will metabolise the auxin, breaking it down and release it into the soil. It will not kill the monocotyledon plants. However, at this concentration, it will kill the dicotyledon plants like dandelion. If you got the concentration wrong and sprayed too much - you would kill the wheat drop. But, if you get it right, you will kill all the weeds, and your crop of wheat, corn, barley etc will be fine.  So having that information is useful for more efficient production of food crops.



11.     Rangitoto College - from wikichat

What are the effects of different forms of auxin on the plants and do differnt forms of auxin effect different parts of a plant?


Karine David, from the seminar
While there are different forms of auxin, they will always trigger the classic growth responses - cell division, phototropism, gravitropism, increased lateral root development etc - but it depends on the concentration. The effect will be different at different concentrations.



12.     Sir Edmund Hilary College - from wikichat
Can Auxins be used to enhance genetically modified (GM) foods?


Karine David, from the seminar

Transgenics  have been used to over-express auxin in a plant. It was done in a tissue specific manner because the scientist were interested in producing seedless fruits. If you remove the seeds from strawberries, you will not get fruit development starting. But if you add auxins to the seedless strawberries the fruit will develop. They chose a promoter that was expressed only in fruits and they managed to produce seedless fruits in strawberry, cucumber, eggplant, tomato, raspberry.