Understanding Plant Growth Optimisation: Case Study

The Work of a Scientific Team

Dr Karine David is group leader within the Plant Molecular Sciences Laboratory at The University of Auckland. This lab has a number of groups looking into different aspects of plant growth and offers excellent training opportunities for young scientists, some of which involve work with Plant and Food Research. Karine’s team are contributing to the worldwide quest for scientific knowledge about HOW auxins work. They are specifically trying to find out more about how the cell can tell that the auxin is there - and then how does the cell “decide” to respond?

As a hormone, auxin will have one (or more) receptor(s) which must recognise the hormone and bind to it, enabling the hormone to act in the cell (see Fig 8). In the case of auxin, that action involves turning on genes that enable plant growth. The fact that auxin has a different effect in different situations (e.g. roots vs shoots) makes understanding this very complex.

In 2005 a group of scientists from the University of Indiana (Dharmasiri et al., 2005) discovered an important part of the mystery around how auxins behave. They identified a protein which was an auxin receptor in the cell and worked out how this receptor was making changes that caused the plant cell to grow. The protein is called Transport Inhibitor Response Protein 1 (TIR1).

In order to grow, growth genes in the cell must be turned on. Normally they are not on - they are repressed by a molecule that binds to the activator. When conditions are favourable for growth, auxin is produced and binds to the TIR1 protein. This Auxin-TIR1 complex turns on a signal to destroy the repressor protein. As a result, the growth genes are turned on (see Fig 9).

Although TIR1 has been shown to be very important, evidence from Dharmasiri et al and others has shown that it is probably not the only auxin receptor. A protein called AUXIN BINDING PROTEIN 1 (ABP1) is also known to bind to auxin and is thought to be able to affect changes in cell expansion. The University of Auckland group, led by Karine, have been investigating the link between Auxin Binding Protein 1 (ABP1) and the responses seen in the plant, trying to find out how the cell responds when auxin and ABP1 are present together.

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Research Questions

How does the cell respond to the presence of the hormone auxin on the receptor site Auxin Binding Protein 1 (ABP1)?
What is the role of Auxin Binding Protein 1 (ABP1) in the cell cycle?

Literature Review

Science is an international, interconnected community. Research starts with a literature review in which the science team find out as much as possible about what is already known that may help them answer the question and design their research method. Key facts that were reported in scientific literature showed that:

ABP1 is an auxin receptor that has been known about for over 30 years (Hertel et al., 1972)
Over-expression of the ABP1 gene (producing more ABP1 than normal) in the tobacco plant allows cells that are normally not responsive to auxins to expand when they are exposed to auxins (Jones et al., 1998)
Mutant plants that make no ABP1 show defective cell elongation, fail to organise the basic plant body plan and die early in development (Napier et al., 2002)
Auxin acts as a “permissive” or starting signal for the cell cycle but little is known about the molecular mechanism that controls this start signal (den Boer and Murray 2000; Stals and Inze, 2001)

The literature also shows that auxins may act directly or indirectly to regulate members of the Cyclin‐dependent Kinase family of substances (John et al., 1993).

The task of the research group was to find out what role ABP1 had in the cell cycle and how the plant “sensed” and responded to auxins via ABP1.

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Research Method

There are a range of methods that scientists can use to find out how a protein behaves. They include:

Over expression - this means there will be more ABP1 in the cell than normal
Down regulation - this means there will be less ABP1 in the cell than normal
Finding a mutant - a mutant that is not producing ABP1 would mean there is no ABP1 in the cell

By comparing the phenotype of normal plants with the phenotype of the plants where ABP1 is either over or under expressed, scientists can build a picture of what the protein ABP1 is responsible for in the plant.

Mutant - Not an Option

With Auxin Binding Protein 1 (ABP1), a mutant that did not make this protein was not an option. Why? Because if you had no ABP1, the plant would not develop. In fact cell division does happen in plants that lack ABP1 due to a mutation, but they show defective cell elongation, fail to organise the basic plant body plan, and will die early in development (Callis, 2005). This means that the mutant was not useful to fully explain the role of ABP1 in the cell cycle. However it did tell scientists that the auxin pathway that regulates cell division was still working in the ABP1 mutant and that ABP1 was necessary for cell elongation and early growth. But it did not explain how.

Chosen Method - Creating Plants That Could be Down Regulated for ABP1

The method decided on was to partially down regulate the ABP1 protein so that there was less ABP1 than normal, but there was some being produced. This was achieved both in cell tissue culture and whole plants using tobacco plants and Arabidopsis (a small fast-growing plant that is ideal for research, see Fig 5). The mechanism used to down regulate ABP1 was to create a transgenic plant that had a gene added that would repress the gene that normally produces ABP1. Exposure of these plants to ethanol vapour was used to turn on this gene, stopping ABP1 production. The tissue culture cells were grown in a broth containing auxin. The plants were grown in growth chambers where all environmental factors could be controlled. Once this was achieved, it was possible to compare the phenotype of the normal cells or plants with the down regulated cells or plants and therefore find out more about the function of the ABP1.

Data Gathering

The growth of the plants and the cells was monitored and observations were made using:

Light microscopy for histological observations
Scanning electron microscopy to enable measurement of cells
Quantitative Real Time Polymerase Chain Reaction (qRT‐PCR) was used to amplify and analyse target DNA molecules to identify whether genes are being expressed or not during the cell cycle

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Findings and Conclusion

The findings show that:

When ABP1 is down regulated (inactivated), plant growth is reduced.
When auxin and auxin binding protein 1 are present together, the cells will grow larger. Auxin binding protein 1 is involved in cell expansion
When auxin binding protein 1 is gradually turned off, less and less CyclinD6 is produced. Given that CyclinD6 is known to control the check point in the cell cycle between Gap1 and DNA Synthesis, this tells us that auxin binding protein 1 is controlling the cell cycle. The same effect was shown for CyclinB1 - the molecule that controls the check point between Gap2 and Mitosis. ABP1 is controlling the cell cycle and therefore cell division.

Discussion

Cell division and cell expansion are two key processes for plant development. Plants develop their organs after germination. Both cell division and cell expansion are important in this process. The research has shown that ABP1 is important in both of these processes. There is, however, a third important component of growth - cell differentiation. This is the process during development where cells specialise.

Future Research

There are still questions that remain unanswered which the research group know would provide useful information to help better understand how plants grow.

They would like to understand all the steps between perception of auxin and the cellular response. This would involve understanding more about gene expression
Having found the role of ABP1 in cell expansion and differentiation, the question to ask is whether ABP1 also has a role in cell differentiation

Direct Application of Knowledge

Knowing how the plant is responding to the presence of auxins is useful in a commercial setting. Karine has a project in which she is collaborating with scientists from Plant and Food to look at control of cell expansion in apples. Cell expansion is directly related to fruit texture. Fruit texture is a complex interaction of many factors such as cell wall chemistry, cell size and shape, cell packing and cell turgor. By potentially enabling control of cell expansion, scientists could develop the ability to control phenotypic features related to fruit texture and therefore increase economic benefit.

Cell Cycle Control

The cell cycle is controlled by molecules called Cyclins. The scientists knew that CyclinD6 controls the check point between Gap 1 and Synthesis in the cell cycle and CyclinB1 controls the check point between Gap 2 and Mitosis.

The scientists wanted to find out whether the genes, CyclinD6 and CyclinB1 were in turn controlled by ABP1.

To answer this question they used real-time PCR to measure the amount of the CyclinD6 or CyclinB1 gene that was being expressed in plants where ABP1 was gradually being down‐ regulated. RT‐PCR is used to show whether target genes (CyclinD6 and CyclinB1) are being expressed in plants.

Fig 15 shows that the more ABP1 was down‐regulated, the less CyclinD1 gene was expressed. This told the scientists that ABP1 was needed for CyclinD1 to do its job in controlling the cell cycle check point. They found similar result for CyclinB1. This information confirmed that if ABP1 was not present, ClyclinB1 and CyclinD6 were not made. This means that ABP1 is controlling the cell cycle by controlling Cyclin production.

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